ABSTRACT The KPC family of serine β-lactamases comprises more than 260 members. Some variants are associated with ceftazidime-avibactam resistance in clinical isolates, often linked to substitutions and/or insertions/deletions (i.e., INDELs) in three distinct loops of the KPC sequence: (i) the 164–179 loop (i.e., the Ω loop); (ii) the 237–243 loop; and (iii) the 267–275 loop. Inconsistencies in residue numbering across published reports, however, complicate the accurate annotation of KPC variants. We retrieved 267 KPC variant sequences from the Beta-Lactamase Database (BLDB) in September 2025 and analyzed sequence differences between variants using combined nucleotide and structure-guided alignment algorithms, supported by AlphaFold3 modeling in ambiguous cases. Variants were classified into four groups to comprehensively review sequence changes across the KPC family: substitutions only ( n = 126), deletions only ( n = 17), insertions only ( n = 66), and variants with two or more types of amino acid changes ( n = 57). Comparisons with previous reports indicate that many annotation errors stem from overlooking the absent residues 58 and 253 in the KPC consensus sequence, and that most inconsistencies in annotation and residue assignment occur in INDEL variants. To address these issues, we propose a standardized annotation scheme for substitutions, deletions, and insertions for the KPC family, based on the Ambler numbering system, and supported by structural information. This systematic scheme will help to standardize the description of newly emerging KPC variants and prevent discrepancies in future reports in the context of antimicrobial resistance.

OBJECTIVE:Plasmid-encoded colistin resistance mcr-1 genes are mainly reported in Escherichia coli worldwide. Here, we characterized a multidrug-resistant Klebsiella pneumoniae 70,241 clinical isolate harbouring mcr-1.1 and blaOXA-181, recovered from a tertiary-care hospital in Tunis, Tunisia. METHODS:Antimicrobial susceptibility was tested by broth microdilution and interpreted using EUCAST 2025 criteria. Whole-genome sequencing was performed with Oxford Nanopore long-read technology. In silico analyses (MLST, resistance genes, plasmid typing) were conducted using tools from the Center for Genomic Epidemiology, while comparative plasmid analysis was performed using BLAST and PROKSEE. Conjugation assays assessed plasmid transferability. RESULTS:K. pneumoniae 70,241 belonged to ST231 and showed a multidrug-resistant phenotype with low-level colistin resistance (MIC = 4 µg/ml). The mcr-1.1 gene was carried on a 33.3-kb IncX4 plasmid closely related to animal- and food-associated plasmids. The blaOXA-181 gene was located within a truncated IS26-based Tn6361-like transposon on a 49.4-kb IncX3 plasmid also harboring qnrS1. Additional plasmids, a fused IncR-IncFIA(HI1) carrying qnrB6, qacE and sul1 and a large IncFIB(K) carrying blaSHV-1 and tet(D), were also found. The mcr-1.1 harbouring IncX4 plasmid was transferred at high frequency (4.6 × 10-2) to E. coli J53, confirming its self-transmissibility. CONCLUSION:This study describes the first genomic features of a K. pneumoniae ST231 clinical isolate from Tunisia co-producing Mcr-1.1 and OXA-181. The presence of highly transmissible IncX4 and IncX3 plasmids highlights the risk of horizontal spread of last-resort resistance genes across clinical and animal settings, emphasizing the need for integrated genomic surveillance within a One Health framework.

Antimicrobial resistance is a silent pandemic responsible for 1.14 million deaths worldwide in 2021, with a major contribution from Klebsiella pneumoniae. β-lactams are the most commonly used antibiotics in humans, and there is an urgent need to characterize the resistance mechanisms to these antibiotics in Enterobacterales other than Escherichia coli. Mecillinam is a narrow-spectrum β-lactam targeting a single penicillin-binding protein, PBP2. Pivmecillinam, its oral prodrug, is used as a first-line treatment for uncomplicated urinary tract infections. It has been used for decades in Europe but was only authorized by the US Food and Drug Administration in 2024. Here, we decipher mecillinam resistance mechanisms in K. pneumoniae by characterizing its resistome in a pan-susceptible strain. The chromosomally encoded SHV β-lactamase led to spontaneous mecillinam-resistant mutants appearing at a higher rate, growing faster and at higher mecillinam concentrations in K. pneumoniae than in E. coli. The most frequent genetic event was an unstable duplication leading to heteroresistance. The selected mutations leading to resistance affected a wide range of functions, with resistance being dependent or independent of the RelA (p)ppGpp synthetase. Through an in-depth characterization of six mutant strains, we showed that, in the presence of mecillinam, they all experienced different growth defects despite high minimal inhibitory concentrations. Overall, our results in K. pneumoniae suggest different mechanisms to escape the complex mode of action of β-lactams in synergy with the β-lactamase SHV.

Putative novel β-lactamase-encoding genes are increasingly identified in whole-genome sequencing (WGS) data, often without phenotypic or biochemical characterization. Here, we characterized the chromosome-encoded GUA-1 Ambler class A β-lactamase from the clinical isolate Pseudomonas guariconensis-like 65411. Among the 40 P. guariconensis genomes available in the GenBank database, only 8 carried a blaGUA-like gene, whereas the others encoded an AmpC β-lactamase, suggesting the existence of two distinct subspecies. Notably, these eight isolates, despite originating from diverse geographical regions, all harbored the blaGUA-like gene inserted at the same chromosomal locus. Heterologous expression in Pseudomonas aeruginosa and Escherichia coli revealed a clavulanic-acid-inhibited cefotaximase-type ESBL. GUA-1 shared 72% amino acid sequence identity with putative Ambler class A β-lactamases from Pseudomonas fulva, Pseudomonas mosselii, and Pseudomonas soli, and 57% with LUT-1, a class A cephalosporinase from Pseudomonas luteola. Kinetic analyses showed that GUA-1 hydrolyzed cefotaxime but not ceftazidime, similarly to LUT-1 and CTX-M-1-type ESBLs. IC50 measurements indicated strong inhibition by clavulanic acid, tazobactam, and avibactam, but not by vaborbactam. Molecular modeling supported these findings, showing favorable binding of cefotaxime in the active site, whereas steric hindrance from Trp274 and electrostatic repulsion from Asp240 likely prevent ceftazidime binding. The blaGUA-1 gene was chromosomally located, with no obvious promoter identified in the immediate upstream region. RT-PCR experiments revealed expression levels comparable to other β-lactamase genes present (blaDHA, blaCMY, blaOXA-1, and blaOXA-204) and to the upstream-located Na+/H antiporter gene. 5' RACE experiments suggested that the blaGUA-1 gene is co-transcribed along with the Na+/H gene. Overall, this study highlights the diversity of β-lactamases within Pseudomonas species.

Resistance to ceftazidime-avibactam (CAZ-AVI) is a growing problem. This study describes the selection of CMY-219, a CMY-42 variant (G156D), conferring resistance to CAZ-AVI in an OXA-484-producing Escherichia coli ST410 after treatment. It raises concern about the risk of selection of CMY variants under CAZ-AVI exposure in ST410 and related clones, which commonly carry CMY-42, are prone to carbapenemase acquisition, and harbor modified PBP3.

Since 2022, the diversity of OXA-48 variants has increased in France. We analyzed the dissemination of five recent variants (OXA-1181, OXA-1201, OXA-1205, OXA-1207, and OXA-1226) mostly identified on plasmids in high-risk Escherichia coli clones. Three of them (OXA-1181, OXA-1205, and OXA-1207) displayed the same Ser244Trp substitution with no significant impact on the β-lactams hydrolytic profile.IMPORTANCEThis study described the recent emergence of five variants (OXA-1181, OXA-1201, OXA-1205, OXA-1207, and OXA-1226) in France. These variants were mostly identified in Escherichia coli high-risk clones. In addition, most of these new OXA-48 variants (OXA-1181, OXA-1205, and OXA-1207) were derived, interestingly, from OXA-181, OXA-232, and OXA-484 variants due to the apparition of the same S244W substitution, a residue that delimits the active site cavity. Together, these results threaten the dissemination of these OXA-48 variants in the community, highlighting the need for continuous monitoring to better understand their epidemiology and dissemination.

Objectives:Carbapenemase-producing Klebsiella pneumoniae (CPKp) represents a major public health threat due to limited treatment options, especially in low- and middle-income countries where colistin often remains the last active antibiotic. Here we investigated clinical carbapenem and colistin-resistant Kp isolates (CCoRKp) from Tunisia between January and June 2023. Materials and methods:Kp isolates were identified by MALDI-TOF, broth microdilution susceptibility testing, NG-Test CARBA5 lateral flow immunoassay, Carba NP test, plasmid analysis and by whole-genome sequencing (WGS) for MLST, genetic relatedness and resistome characterization. Results:Among the 263 Kp isolates collected, 101 (38.4%) were carbapenem resistant and 34 (12.9%) were carbapenem and colistin resistant. Twelve isolates exhibited an extremely drug-resistant phenotype, with in vitro activity retained only for eravacycline and aztreonam/avibactam, two agents that are currently unavailable for clinical use in Tunisia. Nine out of 10 patients who were treated with broad-spectrum antibiotics, including colistin and imipenem, died. WGS revealed OXA-48 and NDM-5 (12/12), CTX-M-15 (9/12) and the 16SRNA methylase ArmA (11/12) and identified ST 101 (n = 5), ST147 (n = 3) and ST 383 (n = 4), suggesting multi-clonal outbreak. While OXA-48 producing ST101 and ST147 were already present in a 2013 collection, ST383 has never been reported in the hospital nor in Tunisia. Conclusion:Here we report double carbapenemase producing and colistin-resistant Kps with limited therapeutic options. Major efforts are needed in infection control and availability of novel molecules in Tunisia to restore safe conditions in hospitals.

ABSTRACT Carbapenemase-producing Enterobacterales (CPEs) have globally emerged and spread beyond human compartments. However, data in wild animals, especially from low- and middle-income countries, such as Algeria, are still very scarce. Here, we investigated CPEs recovered from feces samples collected between October 2021 and June 2023 from wild terrestrial and aquatic mammals, wild migratory/nesters/sedentary birds, and zoo animals, including their environment (water, food, and fecal samples of animal care workers) distributed over six Algerian provinces. Carbapenem-resistant Enterobacterales were characterized using MALDI-TOF-MS, Carba NP, immunochromatographic assay NG-Test CARBA 5, antimicrobial susceptibility testing, and whole-genome sequencing. Thirty CPEs were identified out of the 1,899 samples collected (1.6%). The carriage rate was higher in captive animals (3.2%) than in wild animals (1.2%). Twenty-six produced OXA-48, three OXA-244, and one OXA-181, along with CTX-M-15 ESBL. Clonal expansion of Enterobacter hormaechei hoffmannii ST145 and Klebsiella pneumoniae ST13 was evidenced. Plasmid analysis confirmed that 24/30 isolates harbored a transferable 62 kb IncL pOXA-48 plasmid. Five/six E. coli isolates belonged to high-risk clones with chromosome-mediated blaOXA-244 gene in three isolates, blaOXA-48 in two isolates, and blaOXA-181 gene encoded on an IncFII-ColKP3 hybrid plasmid in one isolate. This study showed widespread dissemination of OXA-48-like producing Enterobacterales in free and captive wild animals, largely driven by epidemic plasmids and clones. It underscores the role of wild animals as a reservoir of CPEs, particularly species living close to humans, such as gulls and pigeons, and occasionally food-producing animals, increasing the risk of bidirectional dissemination between animal, environmental, and human sectors.IMPORTANCEThe global rise of carbapenemase-producing Enterobacterales (CPEs) harboring blaOXA-48-like has been increasingly documented in clinical settings. However, their emergence and transmission in wild and captive animals are less documented. This study provides a high-resolution genomic characterization of CPEs isolated from the feces of wild animals, especially migratory birds, and from captive wild animals, to evaluate the potential risk of dissemination through these animals. Whole-genome sequencing data, genetic investigations, and antimicrobial susceptibility results highlighted the spread of multidrug-resistant CPEs in both animals and humans. The widespread detection of blaOXA-48 across multiple niches suggests sustained circulation beyond hospital settings in Algeria. Human-associated lineages, such as E. coli ST131, ST38, and ST540, were identified with a clear link with humans. This study demonstrates carriage of CPEs in multiple bird species living in areas commonly inhabited by humans and provides further evidence for an effective dissemination of resistance in wildlife, facilitated by feeding habits.

SCOPE:The 2017 European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee report on the role of whole genome sequencing (WGS) in antimicrobial susceptibility testing (AST) concluded that WGS antimicrobial susceptibility prediction (WGS-ASP) was not a sufficiently robust alternative to AST to guide clinical decision making at that stage and that more evidence was required [1]. Since then, the use of WGS, bioinformatic tools, machine learning (ML)/artificial intelligence (AI), databases, and prediction approaches has greatly expanded, along with an increased knowledge of resistance mechanisms and their contribution to antimicrobial susceptibility. In response, a new EUCAST ad hoc subcommittee was established in 2024 to review the literature, with the aim of assessing the current potential and limitations of WGS-ASP. METHODS:As in the previous report, the subcommittee reviewed the literature on a 'by organism' basis but expanded the list to also include enterococci, Haemophilus influenzae, and Bacteroides fragilis in addition to those already included in the first version: Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus pneumoniae, Clostridioides difficile, and Mycobacterium tuberculosis. Additional sections were included to cover advances in metagenomics, other omics technologies and ML/AI. The full report was compiled and reviewed by all subcommittee members before public consultation in November 2025. CONCLUSIONS AND RECOMMENDATIONS:Significant progress has been achieved in WGS-ASP, with growing evidence supporting its ability to distinguish wild-type from non-wild-type isolates and, consequently, susceptible from resistant strains, particularly for M. tuberculosis and when clinical breakpoints align with the epidemiological cut-off (ECOFF). Despite these advances, important challenges remain before WGS-ASP can be adopted as a clinical decision-making tool. Addressing these gaps will require integrated phenotypic and genotypic surveillance to strengthen the evidence base for complex resistance mechanisms and newer antimicrobial agents, alongside comparative assessments that consider both ECOFF and clinical breakpoints. The analyses will require reference method phenotypic AST and high-quality genomic data. It is critical to ensure that datasets reflect the target populations and encompass the full spectrum of antimicrobial susceptibility, while developing unified interpretation frameworks and harmonized bioinformatics tools to standardize outputs. Robust external quality assessment schemes will be essential for clinical validation, and emerging technologies such as AI and ML offer promising avenues to enhance predictive accuracy. Finally, improvements in cost and turnaround time, coupled with evaluations of setting-specific cost-effectiveness, will be key to enabling practical implementation of WGS-ASP.

Background/Objectives: The emergence of plasmid-mediated mcr genes has enabled horizontal dissemination of resistance to colistin, a last-resort antibiotic against multidrug-resistant Enterobacterales. In Tunisia, genomic data on mcr-positive Escherichia coli are still limited. This study reports the genomic characterization of human clinical mcr-positive E. coli isolates from the Military Hospital of Tunis. Methods: Between August 2023 and March 2025, seven E. coli isolates with low-level colistin-resistance (MIC = 4-8 µg/mL) were collected from six patients. They were characterized by antibiotic susceptibility testing and WGS to determine resistome, MLST, genetic relatedness, and plasmid content. Results: The E. coli isolates belonged to diverse sequence types (STs), except for two isolates collected from the same patient 2.5 months apart, which were highly related. Overall, this pattern is consistent with a polyclonal spread. The mcr-1.1 gene was located on IncI2 (n = 5) or IncX4 (n = 2) plasmids, which exhibited high similarity both among themselves and in comparison with plasmids previously reported in human and livestock isolates. Most isolates were multidrug-resistant, harboring acquired resistance genes to multiple antibiotic classes, and chromosomal mutations conferring fluoroquinolone resistance. Three isolates additionally carried chromosomal insertions of the blaCTX-M-55 gene. Resistance to cefiderocol was observed in one isolate and was associated with CirA and Fiu truncation. Conclusions: These findings highlight ongoing dissemination of mcr-1.1-positive E. coli isolates in Tunisia, primarily driven by plasmid transfer. Continuous genomic surveillance and One Health-oriented antibiotic stewardship are essential to limit the spread of colistin-resistance and the emergence of resistance to newer agents such as cefiderocol.

ABSTRACT OXA-48-like carbapenemases have rapidly disseminated worldwide, becoming the most common carbapenemase in many countries, with more than 60 variants reported. Among them, OXA-244 (OXA-48-R214G) and OXA-484 (OXA-181-R214G) are increasingly reported, despite overall reduced hydrolytic activities for β-lactams, including temocillin and carbapenems. R214, located in the β5–β6 loop, through the interaction with D159, is crucial for carbapenem hydrolysis by structuring the active site. R214G variants of OXA-48-likes were analyzed by β-lactam susceptibility testing, steady-state kinetic analyses in the presence or absence of sodium hydrogen carbonate (NaHCO 3 ), molecular modeling, X-ray crystallography, and protein stability assessments using differential scanning fluorimetry (DSF). The R214G substitution in OXA-48 and OXA-181 results in reduced minimal inhibitory concentrations (MICs) for all β-lactams, except for piperacillin and piperacillin/tazobactam combination, for which MICs are increased. OXA-244 and OXA-484 displayed a better affinity for piperacillin with lower K m than for parental enzymes and thus resulted in a higher catalytic efficiency for piperacillin hydrolysis. These results were supported by docking observations, highlighting enhanced affinity when glycine is located at position 214. Overall, DSF indicated that the R214G substitution, while destabilizing the active site, enhances the global stability of the protein, especially for OXA-244. The addition of NaHCO 3 raised the activity and thermal stability of all enzymes, especially of OXA-48, which appeared more sensitive to the presence of NaHCO 3 . Although OXA-48-like R214G is considered a loss-of-function variant, our findings indicate it exhibits increased hydrolytic activity toward piperacillin, which may result in an advantage under piperacillin-tazobactam exposure and thus could contribute to the selection of these globally expanding variants.

BackgroundThe Enterobacter cloacae complex (ECC) includes opportunistic pathogens that can be carbapenem resistant, thus complicating treatment regimens. Here, we characterized a carbapenem- and colistin-resistant ECC O89H7 isolate expressing KPC carbapenemase.Case SummaryECC O89H7 was isolated in January 2021 from an axillary swab performed during routine screening for multidrug-resistant (MDR) bacteria from a patient after 39 days of hospitalization in the intensive care unit (ICU) for severe COVID-19 pneumonia at Nini hospital (Tripoli, Lebanon). The ECC O89H7 was identified as E. roggenkampii (Er) belonging to sequence type (ST)422 and contained eight different plasmids as revealed by whole-genome sequencing (WGS). Er O89H7 was predicted to be a human pathogen (96.3%), harboring 51 virulence factors as well as genes conferring resistance to heavy metals and quaternary ammonium compounds. Er O89H7 was highly drug resistant, including resistance to carbapenems and colistin. The resistome revealed six β-lactamase genes: the chromosome-encoded blaMIR-3 cephalosporinase, blaLAP-2 and blaSHV-12 encoded on a 115-kb IncM-1 plasmid, and blaOXA-10, blaTEM-40, and blaKPC-2 on a mobilizable IncP-6 plasmid of 51 kb, as revealed by mating-out assays.ConclusionHere, we have characterized a human pathogenic Er ST422 harboring blaKPC-2 carbapenemase on an IncP-6 plasmid from Lebanon. Er O89H7 represents a major health threat due to limited therapeutic options, especially because novel β-lactam/inhibitor combinations are not available in Lebanon. Our results highlight an urgent need for improved carbapenemase screening and detection capacity in clinical laboratories and for enhanced genomic surveillance of MDR bacteria to implement intervention strategies to control their spread in Lebanon and beyond.

Genomics have become crucial in addressing the public health challenges posed by antimicrobial resistance (AMR). In this study, we performed the first whole-genome sequencing (WGS) and genomic analyses of clinical Acinetobacter baumannii (A. baumannii) strains isolated at the Sylvanus Olympio University Teaching Hospital in Lomé, Togo. This prospective study, conducted from April 19 to September 02, 2019. Susceptibility profiles were obtained using the Kirby-Bauer disc diffusion method, and the nine studied carbapenem-resistant A. baumannii strains were subjected to next generation sequencing (NGS) using an Illumina platform. All isolates exhibited resistance to imipenem, ticarcillin, clavulanic acid, cefotaxime, and ciprofloxacin, but remained susceptible to colistin, tigecycline, and rifampicin. The study identified five A. baumannii ST1 strains, two ST103 strains, one ST52 strain, and one ST1153 strain. The number of AMR genes per strain ranged from six to 24, whereas the number of virulence genes per strain varied from 32 to 67. Each isolate contained at least one plasmid, with the number of plasmids per isolate ranging from one to four per isolate. The carbapenemase-producing genes blaOXA-23, blaOXA-58, blaOXA-68, blaOXA-69, blaOXA-70, blaOXA-91, and blaNDM-1 were identified, along with blaCTX-M-15 and other antibiotic resistance genes. Additionally, multidrug efflux system genes, including adeCFGHIJKLMNS, abeSJ, and amvA, and a wide array of virulence and biofilm-forming genetic determinants were found in all isolates. Eleven integrons were detected, featuring aac(3)-Ia, sat-2, and dfrA1 cassettes. Tn6018, carrying the mercury resistance gene merR and czcD (Co/Zn/Cd efflux system), and Tn2007, carrying blaOXA-23, were present in six genomes. Four Ghanaian genomes were most closely related to the A. baumannii ST1 and ST103 strains reported in this study. Furthermore, several multidrug resistance plasmids and one virulence and AMR hybrid plasmid (accession number JBFMWK020002174.1) were identified. This study provides valuable insights into clinical A. baumannii in Togo, underscoring the need for more frequent genomic studies in sub-Saharan countries to effectively monitor and combat AMR in Africa.

Background:ESBL-producing Enterobacterales are a major cause of bloodstream infections, requiring rapid detection for timely antimicrobial therapy. We evaluated the performance of the NG-TEST® CTX-M MULTI lateral flow immunoassay combined with the NG-TEST® Blood Culture Prep (BCP) Kit for detecting CTX-M ESBLs directly from positive blood cultures. Methods:In total 165 clinical Enterobacterales were used to spike blood culture bottles. These included 32 non-CTX-M producers, 120 CTX-M producers belonging to the five main CTX-M groups, and 13 Kluyvera sp. Blood cultures were placed in the BACT/ALERT® VIRTUO® system (bioMérieux); upon bacterial growth detection by the system a UriSelect 4 Agar (Bio-Rad) was inoculated to verify purity. Bacteria were prepared using the NG-TEST® BCP Kit followed by the NG-TEST® CTX-M MULTI. Results:The NG-TEST® CTX-M MULTI detected all 120 CTX-M-producing isolates expressing CTX-M variants belonging to the five groups (1, 2, 8, 9 and 25) from both colonies and blood cultures, resulting in 100% sensitivity. The NG-TEST® BCP Kit removed all the patients' blood cells and reliably extracted bacteria irrespective of the bacterial species. Non-CTX-M producers gave negative results, confirming 100% specificity. Among CTX-M-producing Kluyvera species, eight isolates tested positive, while five were repeatedly negative, and consistent with prior reports. Conclusions:Our results demonstrate that the combination of the NG-TEST® BCP Kit and the NG-TEST® CTX-M MULTI systems provides a rapid (<20 min), user-friendly, robust and accurate tool for species-independent detection of CTX-M-type ESBLs directly from positive blood cultures, supporting earlier targeted antimicrobial therapy in bloodstream infections.

OBJECTIVES:The global spread of Klebsiella pneumoniae carbapenemase (KPC)-type carbapenemases has historically been driven by clonal group CG258, but increasing clonal diversification has recently been reported. We aimed to provide a comprehensive phenotypic and genomic characterization of KPC-producing Enterobacterales referred to French National Center (NRC) for Antimicrobial Resistance between 2019 and 2023. MATERIALS AND METHODS:A total of 419 KPC-producing Enterobacterales were referred to the French NRC for Antimicrobial Resistance. Antimicrobial susceptibility testing was performed by broth microdilution. Whole-genome sequencing was conducted for 404 isolates. AMR gene detection, MLST and SNP-based phylogeny were used to analyse resistance mechanisms, clonal structure and outbreak dynamics. RESULTS:KPC-producing Enterobacterales accounted for ∼2% of all carbapenemase-producing Enterobacterales. The K. pneumoniae species complex represented 86% (363/419) of isolates. KPC-3 predominated (328/419, 78%), followed by KPC-2 (87/419, 21%). Additional carbapenemases were detected in 11 isolates. Ceftazidime-avibactam, imipenem-relebactam and meropenem-vaborbactam showed high in vitro activity (97.8%, 95.8% and 97.9% susceptibility, respectively). MLST of 353 K. pneumoniae genomes identified 45 sequence types (STs); ST307 predominated (39.4%) and was associated with a major outbreak in northern France. ST147 (8.8%) and ST512 (7.6%) were also prevalent. Fifteen isolates from ST11, ST39, ST101, and ST395 had high virulence score of 4 (iuc-positive). CONCLUSIONS:KPC-producing K. pneumoniae referred to the French NRC show marked clonal diversification. The emergence of resistant isolates and convergent resistance-virulence profiles underscore the need for sustained genomic surveillance.

Background/Objectives: The global spread of carbapenemase-producing Gram-negative bacteria (CP-GNB) represents a major clinical challenge, causing severe hospital-acquired infections with limited treatment options. Accurate and rapid detection is essential for guiding antimicrobial therapy and implementing infection control measures. Lateral flow immunoassays (LFIAs) targeting the five main carbapenemase families are increasingly used in routine diagnostics, and many new commercial assays have recently become available, often without thorough assessment. The continuous evolution of these enzymes under antibiotic pressure requires regular reassessment of assay performance. Methods: In this study, we evaluated the Beright Carba-5 assay (Alltest Biotech, Hangzhou, China) targeting the five main carbapenemases (KPC, NDM, OXA-48-like, IMP, and VIM), on a panel of 77 whole-genome sequenced Gram-negative bacterial (GNB) isolates exhibiting reduced susceptibility to carbapenems. Seventy-three were carbapenemase-producing (CP) GNBs, including six VIM-, 18 OXA-48-, 14 KPC-, 9 NDM-, 8 IMP-, 10 multiple carbapenemase-, and eight non-targeted carbapenemase-producers. Results: The assay was rapid and easy to use, showing 100% (CI: 73.54% to 100%) specificity, with no false positive results. However, overall sensitivity of CP-GNB detection was lower than expected at 63.08% (CI: 50.20% to 74.72%), with numerous false negatives, particularly among IMP and NDM producers, and to a lesser extent, KPC producers. Detection was more reliable for VIM and OXA-48-like variants. Practical limitations, including insufficient buffer supply, reduced the number of tested isolates from the planned 100 to 77. Conclusions: Overall, the Beright assay demonstrated insufficient sensitivity for routine diagnostic use.

OBJECTIVE:Antimicrobial resistance is a major global health concern, particularly in low- and middle-income countries and conflict-affected settings. This study characterized carbapenem-resistant Gram-negative bacteria (GNB) isolated from clinical samples collected in two Gaza hospitals prior to the 2023 war. METHODS:Between December 2021 and April 2022, GNB from infections were collected at Al Nasser and Al Shifa hospitals. Disc diffusion, synergy, and modified Hodge tests were performed locally. Carbapenemases were confirmed using CarbaNP and NG-Test CARBA 5 assays. Whole-genome sequencing was used to determine the resistome, multi-locus sequence type, and phylogeny. Broth microdilution was used to study antibiotic susceptibility. RESULTS:A total of 135 GNB were recovered, comprising 110 Enterobacterales, 22 Pseudomonas aeruginosa, and 3 Acinetobacter baumannii, predominantly from urine samples (65%). Overall, 87% of the isolates were multidrug-resistant. Meropenem resistance was detected in 100% of A. baumannii isolates, 55% of P. aeruginosa, and 47% of Enterobacterales. Carbapenemase production was confirmed in only 12 isolates: 7 NDM-, 2 VIM-, and 3 OXA-23-producing strains, including 1 OXA-23-NDM co-producing A. baumannii. Whole-genome sequencing of eight isolates revealed one VIM-4-producing Escherichia coli, two NDM-1, one NDM-5, and one NDM-19-producing Klebsiella pneumoniae, and three OXA-23- (including one OXA-23/NDM-1) producing A. baumannii. CONCLUSIONS:Prior to the onset of the war, Gaza hospitals already faced a significant burden of multidrug-resistant and carbapenemase-producing GNB, compounded by limited treatment options and diagnostic challenges. The continued disruption of health care, water, and sanitation systems is likely to accelerate the spread of antimicrobial resistance, underscoring the urgent need for enhanced surveillance and robust antimicrobial stewardship in conflict-affected areas.

The mobile colistin resistance gene, mcr-1, encodes resistance to colistin, a critically important antibiotic. Resistance to colistin can jeopardize antimicrobial chemotherapy. Here, we report the detection and genomic characterization of mcr-1-carrying Escherichia coli isolated from otherwise healthy children in community daycares. The mcr-1 was located on transferable plasmids. Additionally, the mcr-1 occurred in E coli that mainly belonged to a clonal ST10 lineage, indicating that these mcr-1-carrying strains were spreading across daycares in geographically distant cities.

In this work, we demonstrate for the first time the antimicrobial activity of poly-Q binding peptide 1 (QBP1), an anti-amyloidogenic molecule previously identified by phage display for its ability to bind and inhibit the aggregation of polyglutamine proteins like Huntingtin, but also α-synuclein and prion models. Intriguingly, sequence analysis by the ADAPTABLE web server highlighted QBP1's potential antifungal and antibacterial activity, which we have now confirmed experimentally. A theoretical basis for the predicted mechanism of action was provided by molecular dynamics simulations revealing the role of QBP1 aggregates promoting membrane disruption and specific interactions between QBP1 and bacterial and fungal phospholipids, further substantiated by solid-state NMR studies. Using primary human cells, we demonstrated that QBP1 does not display toxicity at high concentrations. Finally, our data demonstrate QBP1's efficacy against some strains of Bacillus cereus, B. mojavensis, Staphylococcus aureus, S. epidermidis, Micrococcus luteus, Enterococcus faecalis, Escherichia coli, and Candida. This unexpected dual function of QBP1 opens new avenues for therapeutic development, potentially restoring the putative antimicrobial protection exerted by many amyloidogenic proteins.

Background Escherichia coli is the primary aetiological agent of urinary tract infections (UTIs), a frequent cause of antibiotic prescription. Over the years, E. coli has become increasingly resistant towards several antimicrobial drugs used to treat UTIs. Objectives To compare the population structures and antimicrobial resistance profiles by molecular characterization of longitudinally collected E. coli from urine samples at two university hospitals in Norway and France. Method Each month in 2019, the first 10 E. coli identified in urine cultures of hospitalized and primary healthcare (PHC) patients were collected at both locations. WGS was performed to detect acquired antimicrobial resistance genes (ARGs) and plasmids and to determine phylogenetic relationships between isolates. Results A total of 478 isolates were included, revealing a high diversity of STs among the isolates. However, the same 10 STs (ST73, ST131, ST69, ST95, ST12, ST141, ST127, ST10, ST58 and ST404) were dominant in both countries, constituting 55.9% (n = 267) of all isolates. Seven of the 10 prevalent STs belonged to phylogroup B2. The number of ARGs varied from 0 (n = 229) to 17 (n = 2). The mean ARG was higher in the French (2.9) than in the Norwegian cohort (2.1, P < 0.001), as well as in the hospitalized (3.0) compared with the PHC cohort (2.0, P = 0.007). Conclusions Despite the differences in patterns of ARGs between the Norwegian and French cohorts, the E. coli population structure was surprisingly highly conserved, suggesting that the distribution of the most common lineages largely depends on factors other than antibiotic use and resistance.