Background/Objectives: Cefiderocol is a novel siderophore cephalosporin with potent in vitro activity against a broad spectrum of Gram-negative bacteria, including carbapenemase-producing Enterobacterales (CPE). However, the recent emergence of resistance in clinical settings raises important concerns regarding its long-term effectiveness. This study aims to investigate the genomic determinants associated with cefiderocol resistance in CPE isolates of human origin. Methods: Comparative genomic analyses were conducted between cefiderocol-susceptible and -resistant CPE isolates recovered from human clinical and epidemiological samples at a tertiary care hospital. Whole-genome sequencing, variant annotation, structural modelling, and pangenome analysis were performed to characterize resistance mechanisms. Results: A total of 59 isolates (29 resistant and 30 susceptible) were analyzed, predominantly comprising Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae. The most frequent carbapenemase gene among the resistant isolates was bla_NDM, which was also present in a subset of susceptible strains. The resistant isolates exhibited a significantly higher burden of non-synonymous mutations in their siderophore receptor genes, notably within fecR, fecA, fiu, and cirA. Structural modelling predicted deleterious effects for mutations such as fecR:G104S and fecA:A190T. Additionally, porin loss and loop 3 insertions (e.g., GD/TD) in OmpK36, as well as OmpK35 truncations, were more frequent in the resistant isolates, particularly in high-risk clones such as ST395 and ST512. Genes associated with toxin–antitoxin systems (chpB2, pemI) and a hypothetical metalloprotease (group_2577) were uniquely found in the resistant group. Conclusions: Cefiderocol resistance in CPE appears to be multifactorial. NDM-type metallo-β-lactamases and missense mutations in siderophore uptake systems—especially in those encoded by fec, fhu, and cir operons—play a central role. These may be further potentiated by alterations in membrane permeability, such as porin disruption and efflux deregulation. The integration of genomic and structural approaches provides valuable insights into emerging resistance mechanisms and may support the development of diagnostic tools and therapeutic strategies.

Backgoround: The genus Pseudomonas encompasses metabolically versatile bacteria widely distributed in diverse environments, including clinical settings. Among these, Pseudomonas kurunegalensis is a recently described environmental species with limited clinical characterization. Objective and Methods: In this study, we report the genomic and phenotypic characterization of a P. kurunegalensis isolate, Pam1317368, recovered from a catheterized urine sample of a post-renal transplant patient without symptoms of urinary tract infection. Initial identification by MALDI-TOF MS misclassified the isolate as Pseudomonas monteilii. Whole-genome sequencing and average nucleotide identity (ANI) analysis (≥95%) confirmed its identity as P. kurunegalensis. The methodology included genomic DNA extraction, Illumina sequencing, genome assembly, ANI calculation, antimicrobial susceptibility testing, resistance gene identification and phylogenetic analysis. Results: Antimicrobial susceptibility testing revealed multidrug resistance, including carbapenem resistance mediated by the metallo-β-lactamase gene VIM-2. Additional resistance determinants included genes conferring resistance to fluoroquinolones and aminoglycosides. Phylogenetic analysis placed the isolate within the P. kurunegalensis clade, closely related to environmental strains. Conclusions: Although the clinical significance of this finding remains unclear, the presence of clinically relevant resistance genes in an environmental Pseudomonas species isolated from a human sample highlights the value of genomic surveillance and accurate species-level identification in clinical microbiology.

Background/Objectives: Cefiderocol is a novel siderophore cephalosporin with potent in vitro activity against a broad spectrum of Gram-negative bacteria, including carbapenemase-producing Enterobacteriaceae CPE). However, the recent emergence of re-sistance in clinical settings raises important concerns regarding its long-term effective-ness. This study aims to investigate the genomic determinants associated with cefiderocol resistance in CPE isolates of human origin. Methods: Comparative genomic analyses were conducted between cefiderocol-susceptible and -resistant CPE isolates recovered from human clinical and epidemiological samples at a tertiary care hospital. Whole-genome sequencing, variant annotation, structural modeling, and pangenome analysis were performed to characterize resistance mechanisms. Results: A total of 59 isolates (29 resistant and 30 susceptible) were analyzed, predominantly comprising Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae. The most frequent car-bapenemase among resistant isolates was blaNDM, which was also present in a subset of susceptible strains. Resistant isolates exhibited a significantly higher burden of non-synonymous mutations in siderophore receptor genes, notably within fecR, fecA, fiu, and cirA. Structural modeling predicted deleterious effects for mutations such as fecR:G104S and fecA:A190T. Additionally, porin loss and loop 3 insertions (e.g., GD/TD) in OmpK36, as well as OmpK35 truncations, were more frequent in resistant isolates, partic-ularly in high-risk clones such as ST395 and ST512. Genes associated with toxin–antitoxin systems (chpB2, pemI) and a hypothetical metalloprotease (group_2577) were uniquely found in the resistant group. Conclusions: Cefiderocol resistance in CPE appears to be multifactorial. NDM-type metallo-β-lactamases and mutations in siderophore up-take systems—especially in the fec, fhu, and cir operons—play a central role. These may be further potentiated by alterations in membrane permeability, such as porin disruption and efflux deregulation. The integration of genomic and structural approaches provides valuable insight into emerging resistance mechanisms and may support the development of diagnostic tools and therapeutic strategies.

Non-tuberculous mycobacteria (NTM) represent an expanding group of environmental microorganisms with growing clinical significance, particularly among immunocompromised patients. In this study, we describe the isolation and comprehensive characterization of a novel Mycobacterium species, strain HUMS_1102779-3, recovered from a sputum sample of a patient undergoing diagnostic bronchoscopy for hilar lymphadenopathy. Initial identification attempts using MALDI-TOF MS and commercial DNA∙STRIP assays were inconclusive, prompting whole-genome sequencing for comprehensive genomic and phenotypic characterization. De novo genome assembly resulted in a 6.2 Mb draft genome with a GC content of 69.03 mol% and high completeness (98.4% conserved orthologues). Phylogenetic and genomic comparisons suggest close relationship to Mycobacterium servetii (ANI 94.52%, dDDH 63.5%), yet below the accepted species delineation thresholds (95% and 70%, respectively), thereby supporting its status as a distinct taxon within the Mycobacterium simiae complex. Phylogenomic reconstruction placed HUMS_1102779-3 within a monophyletic cluster neighboring M. servetii and Mycobacterium helveticum , but genetically divergent from them. The genome encoded 5833 coding sequences, including determinants associated with antimicrobial resistance ( rbpA , rpoB -like variant, mtrA , efpA ), as well as virulence-related genes typical of mycobacteria, such as components of the Type VII secretion system and iron-regulated factors ( ideR , hbhA ). No mobile genetic elements were detected. Phenotypically, the isolate is a fast-growing, scotochromogenic, acid-fast bacillus that exhibits notable dimorphism on 7H11 agar and produce rough yellow–orange colonies after 4–5 days at 35 °C. It differs from M. servetii by several biochemical traits (catalase negative, Tween 80 hydrolysis positive, tellurite reduction-positive) and distinct antimicrobial susceptibility patterns, notably higher MICs for linezolid and minocycline and lower for ciprofloxacin. Based on genomic, phylogenetic, and phenotypic evidence, we propose the designation Mycobacterium cajalii sp. nov.,. The type strain is HUMS_1102779-3 (= CECT 31296 = DSMZ 120455), with the genome deposited under genome assembly accession number ASM4580900v1.

Aeromonas is a genus of Gram-negative, facultatively anaerobic bacteria commonly found in aquatic environments and increasingly recognized as opportunistic pathogens. Among them, Aeromonas dhakensis stands out for its high virulence and antimicrobial resistance, but it is often misidentified due to its phenotypic similarity with A. hydrophila and A. caviae. In this study, a microorganism was isolated from the peritoneal fluid of a patient with signs of intra-abdominal infection. MALDI-TOF MS initially suggested A. hydrophila or A. caviae, but the identification was confirmed with high confidence only after further molecular analyses and the use of genome-based tools, which identified the organism as A. dhakensis. This was further supported by phylogenomic and ANI analysis. Resistome analysis revealed intrinsic resistance genes, including a chromosomal class C β-lactamase and an imiH-type carbapenemase, consistent with the observed carbapenem resistance. No plasmid-mediated carbapenemases were found. These findings underscore the limitations of MALDI-TOF in identifying Aeromonas species in certain cases and highlight the value of genomic approaches for accurate species determination and resistance profiling, especially for isolates from sterile body fluids.