Altogether, 101 OXA-244-Ec isolates from December 2017 to December 2023 were analysed, including 20 isolates from 2017 to 22 and 81 isolates from 2023 (Table 1). These constituted 41.4% of all OXA-48-type-Ec recorded in Poland since 2013 (n = 244) (Figure S1). The OXA-244-Ec isolates were identified in 38 hospitals in 29 cities of 10/16 administrative regions, five of which recorded multiple isolates, including Lubuskie, Pomorskie and Zachodniopomorskie (Fig. 1). Isolates were collected from male (62%) and female (38%) patients, with the mean age of 63.5 years (5 days – 97 years). Most of the isolates (88%) were from colonization (rectal mostly).

Geographic distribution and clonal analysis of OXA-244-Ec isolates in Poland. Geographic distribution of the isolates is shown in the upper part; administrative regions with multiple cases are labelled. Circles represent medical centres where the isolates were recorded. Sizes of the circles are proportional to numbers of cases, and colours refer to individual STs. The dot corresponding to hospital HF1 is transparent in order not to mask other centres in the Lubuskie region. OXA-244-Ec population structure is shown in the bottom part of the figure. The minimum-spanning tree was constructed on the basis of 7-loci MLST data. Each circle represents one ST, and each fragment of a pie chart corresponds to one isolate. The size of a circle is proportional to the number of isolates of this ST. Connecting lines infer phylogenetic relatedness in terms of allelic differences; thick solid line indicates a single-locus variant

Fourteen STs were discerned using the mlst tool (https://github.com/tseemann/mlst) (Table 1; Fig. 1). ST38 dominated (n = 70; 69.3%), and was followed by ST58 (n = 9; 8.9%), ST10 (n = 4; 4.0%), ST69, ST167 and ST361 (n = 3; 3% each), and ST131 (n = 2; 2%). The remaining STs had single isolates. ST38 comprised the first organism identified retrospectively, being a sporadic isolate from Cracow, December 2017. By the end of 2023, OXA-244-Ec ST38 was recorded in 19 hospitals in 18 towns of eight regions (Table 1; Fig. 1). Lubuskie with the outbreak hospital HF1 [7] was the most affected area (n = 55; 78.6% of ST38). The clonal diversity of OXA-244-Ec has been observed also in other countries [2, 3, 5].

The 70 ST38 isolates were subjected to SNP-based comparative analyses, using BioNumerics v.7.6.3 (Applied Maths NV, Sint-Martens-Latem, Belgium) with a strict SNP filtering template. The in-sample clonality analysis utilised the first HF1 outbreak isolate (isolate 5550/23) [7] as a reference. The phylogenetic analysis was done against all 164 OXA-244-Ec ST38 genomes available in GenBank (as of August 27, 2023; selected from 217,882 E. coli genomes) and 100 OXA-244-Ec ST38 genomes described in Germany [2]. The in-sample analysis revealed 6890 polymorphic positions within 4.3 Mb (78%) of the reference genome, and SNP numbers between any isolate and the reference was 0-3334 SNPs (Table 1). Despite this overall diversity, the majority of the isolates (n = 53; 75.7% of ST38; all from 2023) formed a tight cluster with 0–9 SNPs between members, representing the on-going outbreak in Lubuskie [7]. The epicentre HF1 recorded eight further isolates in July-October 2023 (n = 47; 67.1% in total) and exported the organism in June-November 2023 (n = 7) to several hospitals in the same and the neighbouring region (Table 1; Fig. 1). Four outbreak cases in these new hospitals had been treated previously (1–6 months ago) in HF1. As it was reported, the epidemic organism was classified into the European OXA-244-Ec ST38 Cluster B, marked by the extended-spectrum β-lactamase (ESBL) gene blaCTX−M−14 (Fig. 2) [1, 7]. Five other Cluster B isolates, recovered in 2018-23 in several regions, had no links to the outbreak, being distant by 1073–2979 SNPs from the reference. In the phylogenetic analysis all Cluster B isolates, including the Lubuskie outbreak, were split into five branches of the highly varied lineage, shared mostly with isolates from Germany, The Netherlands and the UK (Fig. 2).

Single-nucleotide polymorphism (SNP)-based phylogenetic tree of OXA-244-Ec ST38 isolates from Poland (n = 70), GenBank (n = 164; all international isolates as of August 27, 2023) and Germany (n = 100) [2]. Numbers on the inner circle are the original numbers of the study isolates, GenBank assembly numbers and original numbers from the German study. Country symbols: AU, Australia; CA, Canada; CO, Colombia; CZ, Czechia; DE, Germany; FR, France; GB, United Kingdom; NL, the Netherlands; NO, Norway; PL, Poland; QA, Qatar; TR, Turkey; US, USA. The outbreak isolates are marked with dark gray, whereas the remaining Polish isolates with light gray. Coloured squares represent selected AMR genes. The tree was constructed using Parsnp and visualised with iTOL

The second ST38 fraction of isolates (n = 12; 17.1%) occurred in nine towns of five regions in 2017-23 (Fig. 1). Classified into the Cluster A [1], these had the marker blaCTX−M−27 ESBL gene and were notably homogeneous, differing only by 0–44 SNPs from each other (Table 1; Fig. 2). A set of four isolates with 0–1 SNPs was discerned, representing an outbreak in hospital HZ1, region Zachodniopomorskie (Table 1; Fig. 1). These were cultured in five days from three newborns (5–9 days) and one female patient (23 years) in one obstetrics and gynaecology ward. The Cluster A isolates were scattered across the large and homogeneous branch of the OXA-244-Ec ST38 phylotree, and their closest relatives were from Germany, The Netherlands, Norway and France. (Fig. 2).

Another important clone was ST58, with nine isolates from 2023, collected in four centres of three cities and regions, mostly Gdańsk in Pomorskie. Seven isolates from hospitals HG1 and HG2 showed 0–2 SNPs in between, indicating outbreak in HG1 with transmission to HG2. OXA-244-Ec ST58 has been rare, with only single, unrelated isolate from 2015 from Egypt (GenBank acc. No. GCA_024389345.1).