Francisco González-Espinosa

Here, we described the resistome and virulome of worldwide K. pneumoniae ST25 (Kp-ST25) genomes and established their phylogenetic relationship. Out of a collection of 32 KPC-2-producing Kp-ST25 recovered in Argentina during 2015–2017, eight isolates were selected for whole-genome sequencing (WGS). Additionally, 256 Kp-ST25 genomes were downloaded from NCBI. Bioinformatic analysis was carried out using Unicycler, PROKKA, and Quast. Resistome and virulome were inspected using Kleborate, AMRFinderPlus, BACMET, and VFDB. Phylogenetic estimation included a core genome SNP-based analysis carried out using Roary and IQ-TREE. The tree was visualized using Microrreact. The capsular type K2 was detected in 98% of Kp-ST25 genomes. LPS O1 ab was the most prevalent, followed by O2 afg and O3/O3 a . Most isolates carried the yersiniabactin coding gene, while aerobactin, salmochelin, and RmpADC coding genes were less common. Resistome includes multiple genes for aminoglycosides and quinolones resistance, while 23% of Kp-ST25 harbored carbapenemase-encoding genes ( bla _KPC-2,-3 ; bla _NDM-1,-4,-7 ; bla _{OXA-48,-181,-232} ). In 37% of Kp-ST25, ESBL-encoding genes ( bla _CTX-M-2,-15 , bla _SHV-12 ) were detected as the only acquired bla marker. Phylogeny revealed two clades. Clade A is smaller and more virulent, and Clade B is larger and more diverse in virulence and resistance features. Argentinian isolates fall within clade B, showing high resistance and moderate virulence, except one isolate, which displays higher virulence. Furthermore, the Kp-ST25 lineage exhibited intermediate virulence and resistance levels in the genomic analysis. IMPORTANCE: Hypervirulent K. pneumoniae (hv Kp ) strains cause invasive infections in both healthy and immunocompromised individuals, displaying a more virulent phenotype than classical strains. Apart from lipopolysaccharides (LPSs) and fimbriae, hv Kp can present a hypermucoid phenotype due to K1, K2, or K5 polysaccharide capsule and acquired virulence factors encoding the siderophores yersiniabactin (Ybt), aerobactin (Iuc), and salmochelin (Iro), as well as the genotoxin colibactin (Clb). Furthermore, hv Kp is becoming increasingly resistant to antibiotics, rendering infection by these strains very challenging to treat. Most of the hv Kp isolates belong to sequence types ST23, ST25, ST65, and ST86. Among these, K. pneumoniae ST25 was first detected in Argentina in a surveillance study during 2015–2017. These isolates displayed resistance to carbapenems, one of the last-resort antibiotics for Gram-negative complicated hospital infections. The present study contributes to the knowledge of the virulence, resistance, and phylogeny of K. pneumoniae ST25 isolated in Argentina, along with others recovered worldwide.

Background: Carbapenem-resistant Enterobacterales (CRE) are a global public health concern, with carbapenem-resistant Klebsiella pneumoniae (CR-Kp) recognised as the highest-priority pathogen. This study aimed to investigate the epidemiological features of CRE isolates throughout the COVID-19 pandemic in Buenos Aires, Argentina. Methods: A prospective study was conducted in two hospitals from 2019 to 2022, recovering all CRE from inpatients. Antimicrobial susceptibility was performed by automated and/or manual tests, according to CLSI. β-lactamases detection was performed using Multiplex PCR and MALDI-TOF MS. Kp typing was assessed by multiplex PCR and/or MLST based on WGS. Results: 22% (359/1594) were CRE, predominantly CR-Kp. Overall, high non-susceptibility (NS) rates were observed in both centres. NS remained largely stable in HA, except for a significant increase in colistin NS, whereas HB showed a rise in NS to multiple antimicrobials over time. A significant shift from multidrug-resistant to extensively drug-resistant and difficult-to-treat phenotypes was observed across the study periods. Out of 359 CRE, bla_KPC was confirmed in 141, bla_NDM in 170, and bla_KPC + bla_NDM in 20 isolates. Before the COVID-19 pandemic, KPC was the main carbapenemase in HB, while NDM was already the prevalent one in HA. In 2022, both enzymes showed similar prevalence. bla_KPC-2 and bla_NDM-5 were the prevalent alleles in K. pneumoniae. Before the COVID-19 pandemic, K. pneumoniae epidemiology varied by hospital, characterised by clonal diversity; however, in 2022, CG258-tonB79 drove the epidemiology in both hospitals. Conclusions: A more extensive resistance phenotype among CRE was evidenced throughout the COVID-19 pandemic, driven by carbapenemase-producing K. pneumoniae. NDM-5 and KPC-2 were the main carbapenemases identified. A temporal shift in carbapenemase prevalence was observed in each hospital, converging in similar frequencies of KPC and NDM by 2022 across both centres. This scenario was driven by the active dissemination of K. pneumoniae ST258.

Achromobacter spp. are intrinsically resistant to multiple antibiotics and can also acquire resistance to those commonly used for the treatment of respiratory infections, especially in patients with cystic fibrosis. The aim of this study was to perform the genetic and biochemical characterization of AXC-2 from A. ruhlandii and to analyze all available AXC variants. Steady-state kinetic parameters were determined on a purified AXC-2 enzyme. It exhibited higher catalytic efficiencies towards amino-penicillins and older cephalosporins, while carbapenems behaved as poor substrates. Phylogenetic analysis of all bla_AXC variants available in the NCBI was conducted. AXC was encoded in almost all A. ruhlandii genomes, whereas it was only found in 30% of A. xylosoxidans. AXC-1 was prevalent among A. xylosoxidans. AXC variants were clustered in two main groups, correlating with the Achromobacter species. No association could be established between the presence of bla_AXC variants and a specific lineage of A. xylosoxidans; however, a proportion of AXC-1-producing isolates corresponded to ST 182 and ST 447. In conclusion, this study provides valuable insights into the genetic context and kinetic properties of AXC-2, identified in A. ruhlandii. It also provides a thorough description of all AXC variants and their association with Achromobacter species and various lineages.