Juan José González-López

Introduction: Pseudomonas is one of the largest genera among Gram-negative bacteria, currently comprising nearly 400 valid species. Among them, non- aeruginosa Pseudomonas species, together with Stutzerimonas —a genus recently separated from Pseudomonas —are becoming important reservoirs of antimicrobial resistance genes. However, accurate species-level identification of these organisms remains challenging when relying on the methods routinely implemented in most clinical microbiology laboratories, such as MALDI-TOF mass spectrometry. Therefore, this study aimed to identify and characterize metallo-β-lactamase-producing non- aeruginosa Pseudomonas spp. and Stutzerimonas spp. isolates obtained in a Spanish tertiary hospital, focusing on their species-level identification, dissemination and role as reservoirs of carbapenemase-encoding genes. Methods: Whole-genome sequencing (WGS) and antimicrobial susceptibility testing (AST) were used to identify and characterize a total of 22 carbapenemase-producing non- aeruginosa Pseudomonas and Stutzerimonas isolates collected from patient and environmental samples from March 2022 to July 2024 in a Spanish tertiary hospital. Results: WGS analysis identified eight species: Pseudomonas alloputida ( n = 7), Pseudomonas mosselii ( n = 5), Pseudomonas kurunegalensis ( n = 4), Pseudomonas asiatica ( n = 2), Pseudomonas oleovorans ( n = 1), Pseudomonas soli ( n = 1), Stutzerimonas chloritidismutans ( n = 1) and Stutzerimonas stutzeri ( n = 1). Multilocus sequence typing (MLST) classified isolates of the P. putida group into nine STs, revealing three predominant clones: P. alloputida ST69, P. mosselii ST115 and P. kurunegalensis ST114. AST results showed resistance rates >55% to all tested β-lactams, aminoglycosides and fluoroquinolones, except for cefiderocol, amikacin and colistin (0–5%). The following carbapenemase combinations were detected: bla _VIM−1 ( n = 16), bla _VIM−1 + bla _VIM−2 ( n = 4), bla _VIM−1 + bla _NDM−1 ( n = 1) and bla _VIM−11 ( n = 1). Seven distinct bla _VIM -harboring plasmids were detected in 16 isolates, whereas the remaining six carried chromosomally-integrated bla _VIM . Eight different bla _VIM -harboring class I integrons were detected, two of which also occurred in P. aeruginosa clinical isolates obtained in the same hospital. Conclusion: This study highlights the epidemiological relevance of non- aeruginosa Pseudomonas and Stutzerimonas species as reservoirs of metallo-β-lactamase genes encoded in mobile genetic elements. These findings reinforce the need for systematic WGS-based surveillance of both clinical and environmental isolates, enabling the detection of hidden transmission networks and interspecies dissemination of antimicrobial resistance determinants.

Neisseria meningitidis clonal complex 11 (cc11) is a hyperinvasive lineage associated with global dissemination and outbreaks, including the ‘South American/UK’ sublineage. In Spain, cc11 serogroup W cases have increased in recent years; however, the evolutionary dynamics of circulating variants remain insufficiently characterized. This study aimed to assess the clonal diversity, temporal evolution and antimicrobial resistance profiles of cc11 isolates recovered in Spain between 2011 and 2025. A total of 1,226 isolates (406 invasive and 820 non-invasive) were collected across 8 Spanish regions, from which cc11 isolates were selected for analysis. Whole-genome sequencing (WGS) was used to perform core-genome multilocus sequence typing and SNP-based Bayesian phylogenetics to characterize population structure. Antimicrobial susceptibility was assessed using gradient diffusion, and genetic determinants of resistance were identified by WGS. We identified 179 cc11 isolates, mostly belonging to genogroup W (73.7%) and lineage 11.1 (85.5%). The ‘South American/UK’ sublineage was the main sublineage and displayed extensive diversification. A previously undescribed variant, designated the ‘2020 strain’, emerged with an estimated most recent common ancestor in 2017 and accounted for 77.2% of ‘South American/UK’ sublineage isolates recovered between 2023 and 2025. This variant was mainly identified in respiratory samples from men who have sex with men but was also associated with invasive meningococcal disease in other population groups. The ‘2020 strain’ exhibited resistance to penicillin and azithromycin, likely driven by penicillin-binding protein 2 and MtrCDE alterations. The emergence of this antimicrobial-resistant variant highlights the adaptive capacity of cc11 and the importance of genomic surveillance to monitor its evolution and public health relevance.

Purpose: To describe the first outbreak of KPC-3-producing Klebsiella pneumoniae ST512 in Aragón, Spain, and characterize its clinical, microbiological, and genomic features, including plasmid dynamics, resistance mechanisms, and phylogenetic context. Methods: Between July 2022 and July 2024, 130 KPC-3-producing K. pneumoniae isolates were recovered from 33 patients during an outbreak at a tertiary-care hospital in Zaragoza. Antimicrobial susceptibility testing and whole-genome sequencing were performed. Phylogenomic (SNP and cgMLST) and plasmid analyses defined clonal relatedness and plasmid structures. Comparative genomics with 985 international ST512/KPC-3 genomes determined phylogeographic relationships. Results: Most cases (84.6%) were detected through active surveillance. All the isolates were resistant to β-lactams, ceftolozane/tazobactam, tobramycin and amikacin, while 64.4% remained susceptible to gentamicin. All the isolates were susceptible to cefiderocol, colistin, and tigecycline. One ceftazidime/avibactam-resistant isolate carrying bla _KPC−70 emerged after prolonged therapy. Genomic analysis confirmed a clonal outbreak of Klebsiella pneumoniae ST512 (≤ 16 SNPs; ≤13 cgMLST allelic differences). Phylogenetic comparison showed that the isolates were genetically close to those from Italy and central Spain. All isolates carried bla _KPC−3 within Tn 4401b . Three bla _KPC−3 plasmid structures were identified: an IncFII(K) plasmid (pHCUKPC3), a ColEST258 variant, and a novel cointegrate plasmid (pHCUKPC3co). The virulence-associated factors identified included yersiniabactin (ybt10/ICE Kp4 ), KL107 capsular type, and O2afg O-antigen. Conclusion: This study documents the wider dissemination of ST512/KPC-3 as a high-risk clone in Spain, characterized by persistence driven by clonal dissemination, selective pressure, and plasmid plasticity. Our findings highlight the need to integrate genomic and plasmidomic surveillance to anticipate resistance evolution and contain high-risk clones.

BACKGROUND: Dual penicillin- and ciprofloxacin-resistant Neisseria meningitidis causing invasive meningococcal disease (IMD) have recently emerged in association with sequence type (ST) 3587, harbouring ROB-1 β-lactamase ( bla _ROB-1 ) and a mutated DNA gyrase ( gyrA ). These strains pose a threat to current antimicrobial treatment and prophylaxis. AIM: We aimed to characterise the first dual-resistant N. meningitidis ST-3587 isolates harbouring bla _ROB-1 and a mutated gyrA identified in Spain. METHODS: Three N. meningitidis isolates encoding bla _ROB-1 were identified in 2024. They were characterised by whole genome sequencing to determine capsular genogroups, ST and genetic antimicrobial resistance markers. Dated phylogenetic analysis was performed alongside global ST-3587 strains. RESULTS: The three bla _ROB-1 -encoding isolates belonged to ST-3587, genogroup Y, harboured a T91I mutation in gyrA and showed resistance to penicillin and ciprofloxacin. These isolates were obtained from urethral, oropharyngeal and blood samples, each from a different patient. According to the dated phylogenetic analysis of ST-3587 and the presence of bla _ROB-1 , two clades were defined: clade I and clade II. Within clade II, subclade II.I was identified, comprising isolates which, in addition to bla _ROB-1 , carried the T91I mutation in gyrA . This subclade included the three Spanish isolates, which exhibited close genetic relatedness. CONCLUSION: This study documents the emergence of N. meningitidis ST-3587 with dual resistance in Europe, including a documented urogenital infection by this lineage. Continued surveillance of antimicrobial resistance in N. meningitidis , including non-invasive cases, is crucial for timely public health responses and effective IMD prevention strategies.

Introduction: Patients with extremely elevated IL-6 levels remain poorly characterized, and no specific plasma concentration has been established to reliably predict mortality or guide immunomodulatory interventions. We hypothesized that extreme hypercytokinemia is associated with increased mortality in sepsis. The primary objective was to identify, in patients with hyperinflammatory endotype, an IL-6 threshold associated with a significantly elevated risk of death. Methods: We conducted a retrospective, single-center observational study based on a historical cohort of adult patients with consecutive activation of the in-hospital sepsis code, a prospective and standardized institutional care pathway, at Vall d’Hebron University Hospital between July 2018 and December 2024. Patients fulfilling Sepsis-2 diagnostic criteria and criteria for severe sepsis or septic shock were eligible. Plasma interleukin-6 (IL-6) levels were routinely determined in all patients. The analysis included patients with complete clinical and laboratory data available in the study database. To identify the IL-6 threshold associated with critical risk of death, a cumulative conditional relative frequency analysis was performed. A quantile-based analysis was conducted using predefined intervals of 1000 pg/mL and 15,000 pg/mL. A multivariable logistic regression analysis was conducted to identify clinical and laboratory parameters independently associated with IL-6 > 15,000 pg/mL and outcome. Results are presented as odds ratios (ORs). Survival differences were assessed using Kaplan–Meier analysis. Results: Overall mortality was 31% in the 1669 patients analyzed. Median IL-6 concentration was 772 pg/mL (IQR: 164–8750 pg/mL) with significantly higher levels in non-survivors (2137 pg/mL, IQR: 267–34,758). A critical IL-6 cutoff of 14,930 pg/mL was identified, which was rounded to 15,000 pg/mL for clinical applicability. IL-6 > 15,000 pg/mL was associated with increased mortality (OR 2.22, 95% CI: 1.12–5.36). Kaplan–Meier analysis revealed significantly reduced survival in patients above this IL-6 threshold (p < 0.0001). Conclusions: In this cohort of patients with severe sepsis or septic shock, plasma IL-6 levels > 15,000 pg/mL defined a critical threshold beyond which mortality risk exceeded survival probability. Critical hypercytokinemia may serve as a clinically relevant biomarker to identify patients with sepsis and multiorgan dysfunction who could benefit from precision immunomodulatory therapies.

Pathogens adapting to the human host and to vaccination-induced immunity may follow parallel evolutionary paths. Bordetella parapertussis ( Bpp ) contributes significantly to the burden of whooping cough (pertussis) and shares vaccine antigens with Bordetella pertussis (Bp) ; both pathogens are phylogenetically related and ecological competitors. Bp vaccine antigen-coding genes have accumulated variation, including pertactin (PRN) disruptions, after the introduction of acellular vaccines in the 1990s. We aimed to evaluate evolutionary parallelisms in Bpp , even though pertussis vaccines were designed against Bp . We sequenced 242 Bpp isolates collected in France, the USA and Spain between 1937 and 2019, spanning pre-vaccine and two vaccines eras. We investigated the temporal evolution of Bpp sublineages using a Bayesian approach based on whole-genome SNPs and performed comparative genomic analyses focusing on antigen and virulence gene loci. The most recent common ancestor of all sequenced Bpp isolates was estimated around the year 1877, making it one of the youngest human pathogens, and the Bpp evolutionary rate we estimated (2.12×10 ⁻⁷ substitutions per site per year) was remarkably similar to the one previously reported for Bp (2.24×10 ⁻⁷ ). PRN antigen deficiency in Bpp was driven by 18 disruptive mutations, including deletion prn :ΔG-1895 estimated to have occurred around 1998 and observed in 73.8 % (149/202) of post-2007 Bpp isolates. In addition, we detected two early (year ~1900) mutations in the bvg A- fhaB intergenic region, which controls multiple virulence factors including the filamentous haemagglutinin antigen. Gene clusters for pertussis toxin and fimbriae showed a surprising lack of gene decay. Our findings suggest early adaptation of Bpp to humans through modulation of the bvgAS regulon, and a rapid adaptation through the loss of PRN expression, representing a late evolutionary parallelism concomitant with acellular vaccination against whooping cough.

Invasive meningococcal disease (IMD) is associated with significant global morbidity and mortality and is addressed by conjugated polysaccharide and subcapsular vaccines. In Spain, data on 4CMenB vaccine strain coverage and antimicrobial susceptibility are limited. This study aimed to describe the genomic epidemiology, predict 4CMenB vaccine strain coverage, and assess antimicrobial susceptibility of 323 Neisseria meningitidis isolates causing IMD, collected from 57 Clinical Microbiology Laboratories in Spain over 12 years (2011/12-2022/23). Whole genome sequencing was performed to identify serogroup, clonal complex (cc), and antimicrobial resistance determinants. Vaccine strain coverage for serogroup B (MenB) isolates was predicted using the genetic Meningococcal Antigen Typing System approach. The most prevalent serogroups were B (57.9%), W (21.4%), C (10.4%), and Y (8.4%). MenB predominated throughout most seasons, except during the 2019/20 season when serogroup W peaked. Post-COVID-19 pandemic, MenB remained the most frequent (70.2%). Thirteen cc were identified among MenB isolates, with cc213 being the most prevalent (40.1%). Only 28.9% of MenB isolates were predicted to be covered by 4CMenB, with cc213 showing an exceptionally low coverage rate (5.3%) due to antigenic variants poorly targeted by the vaccine. Notably, cc213 was responsible for twice the proportion of MenB cases in 4CMenB-vaccinated versus unvaccinated. All isolates were susceptible to third generation cephalosporins, and 13.5% showed penicillin resistance. This study highlights the alarming prevalence of cc213 among MenB IMD cases in Spain and the limited 4CMenB coverage against this cc. The disproportionate representation of cc213 in vaccinated individuals underscores its potential to compromise vaccine effectiveness.