Background. The use of antibiotics in food production selects for resistant bacteria and may cause a threat to human and animal health. Belgium and the Netherlands have one of the highest densities of broilers and pigs in Europe, making active monitoring of antibiotic use and resistance in this region vital. Objectives. This study aimed to quantify ESBL-producing (ESBL-E. coli), carbapenem- and ciprofloxacin-resistant (CiproR) Escherichia coli in animal feces on broiler and pig farms with a history of high antibiotic use in Belgium and the Netherlands. Methods. A total of 779 broiler and 817 pig fecal samples, collected from 29 conventional broiler and 31 multiplier pig farms in the cross-border region of Belgium and the Netherlands, were screened for the presence of antibiotic-resistant E. coli using selective culturing. Results. Carbapenem-resistant E. coli were not detected. ESBL-E. coli were remarkably more prevalent in samples from Belgian than Dutch farms. However, CiproR-E. coli were highly prevalent in broilers of both countries. The percentage of samples with ESBL- and CiproR-E. coli was lower in pig compared to poultry farms and varied between farms. No clear association with the on-farm antibiotic use in the year preceding sampling was observed. Multidrug resistance was frequently observed in samples from both countries, but ESBL-production in combination with ciprofloxacin resistance was higher in samples from Belgium. Conclusions. This study demonstrated marked differences in antibiotic resistance between countries, farms and within farms. The observed variation cannot be explained straightforward by prior quantity of antibiotic use suggesting that it results from more complex interactions that warrant further investigation.

Whole-genome sequencing (WGS) is becoming the de facto standard for bacterial typing and outbreak surveillance of resistant bacterial pathogens. However, interoperability for WGS of bacterial outbreaks is poorly understood. We hypothesized that harmonization of WGS for outbreak surveillance is achievable through the use of identical protocols for both data generation and data analysis. A set of 30 bacterial isolates, comprising of various species belonging to the Enterobacteriaceae family and Enterococcus genera, were selected and sequenced using the same protocol on the Illumina MiSeq platform in each individual centre. All generated sequencing data were analysed by one centre using BioNumerics (6.7.3) for (i) genotyping origin of replications and antimicrobial resistance genes, (ii) core-genome multi-locus sequence typing (cgMLST) for Escherichia coli and Klebsiella pneumoniae and whole-genome multi-locus sequencing typing (wgMLST) for all species. Additionally, a split k-mer analysis was performed to determine the number of SNPs between samples. A precision of 99.0% and an accuracy of 99.2% was achieved for genotyping. Based on cgMLST, a discrepant allele was called only in 2/27 and 3/15 comparisons between two genomes, for E. coli and K. pneumoniae, respectively. Based on wgMLST, the number of discrepant alleles ranged from 0 to 7 (average 1.6). For SNPs, this ranged from 0 to 11 SNPs (average 3.4). Furthermore, we demonstrate that using different de novo assemblers to analyse the same dataset introduces up to 150 SNPs, which surpasses most thresholds for bacterial outbreaks. This shows the importance of harmonization of data-processing surveillance of bacterial outbreaks. In summary, multi-centre WGS for bacterial surveillance is achievable, but only if protocols are harmonized.

Multidrug-resistant Acinetobacter baumannii is a major concern in healthcare institutions worldwide. Several reports described the dissemination of A. baumannii high-risk clones that are responsible for a high number of difficult-to-treat infections. In our study, 19 multidrug-resistant A. baumannii strains from Budapest, Hungary, were investigated based on whole-genome sequencing (WGS). The obtained results were analysed together with data from 433 strains of A. baumannii from the Pathogenwatch database. WGS analysis of 19 A. baumannii strains detected that 12 belonged to ST2 and seven belonged to ST636. Among ST2 strains, 11 out of 12 carried either bla_OXA-23 or bla_OXA-58 genes; however, all strains of ST636 uniformly carried bla_OXA-72 gene. All strains of ST2 and ST636 carried bla_OXA-66 and bla_ADC-25 genes. Based on core genome multilocus sequence typing (cgMLST), 10 strains of ST2 belonged to cgMLST906, one strain to cgMLST458, and one strain to cgMLST1320; by contrast, all strains of ST636 belonged to cgMLST1178. Certain virulence determinants were present in all strains of both ST2 and ST636, namely, Ata, Bap, BfmRS, T2SS and PNAG. Interestingly, OmpA was present in all strains of ST2, but it was absent in all strains of ST636. Comparative analysis of 19 strains of this study and the collection of 433 isolates from Pathogenwatch database, proved a diverse clonal distribution of high-risk A. baumannii clones in Europe. The major clone in Europe is ST2, which is present all over the continent. However, ST636 has been mainly reported in Eastern Europe. Interestingly, cgMLSTs of ST2 correspond to the production of different beta-lactamases, namely, OXA-82 in cgMLST116, OXA-72 in cgMLST506, and cgMLST556, PER-1 in cgMLST456 and cgMLST1041. Our study demonstrates that the ST2 high-risk clone of A. baumannii is the most widespread in Europe; however, based on cgMLST analysis, a detailed detection of beta-lactamase production can be determined.

Klebsiella pneumoniae is an opportunistic pathogen known for two main pathotypes: classical K. pneumoniae (cKp), often multidrug-resistant and common in hospitals, and hypervirulent K. pneumoniae (hvKp), associated with severe community-acquired infections. The recent emergence of strains combining hypervirulence and resistance is alarming. This study investigates the distribution of sequence types (STs), resistance, and virulence factors in K. pneumoniae strains causing bloodstream and urinary tract infections in Croatia. In 2022, 200 consecutive K. pneumoniae isolates were collected from blood and urine samples across several Croatian hospitals. Whole genome sequencing was performed on 194 isolates. Within the analyzed K. pneumoniae population, the distribution of sequence types was determined with multi-locus sequence typing (MLST) and capsule loci, resistance, and virulence determinants were assessed with the bioinformatics tool Kleborate. The analysis identified 77 different STs, with ST101 (24.6%) being the most prevalent, predominantly linked to the K17 capsular type (CT), invasive device usage, high antimicrobial resistance, and low virulence scores. The highest virulence scores were recorded in ST86 isolates, which were predominantly linked to the K2 CT and included some strains with medium resistance scores. String tests were positive in 19 strains, but only four of those harbored hypermucoviscous genetic determinants. The most prevalent ST101 clone in Croatia demonstrated a diverging association between resistance and virulence. An alarming co-existence of resistance and virulence was recorded in the ST86 strains.

Background: Colistin serves as the last line of defense against multidrug resistant Gram-negative bacterial infections in both human and veterinary medicine. This study aimed to investigate the occurrence and spread of colistin-resistant Enterobacterales (ColR-E) using a One Health approach in Belgium and in the Netherlands. Methods: In a transnational research project, a total of 998 hospitalized patients, 1430 long-term care facility (LTCF) residents, 947 children attending day care centres, 1597 pigs and 1691 broilers were sampled for the presence of ColR-E in 2017 and 2018, followed by a second round twelve months later for hospitalized patients and animals. Colistin treatment incidence in livestock farms was used to determine the association between colistin use and resistance. Selective cultures and colistin minimum inhibitory concentrations (MIC) were employed to identify ColR-E. A combination of short-read and long-read sequencing was utilized to investigate the molecular characteristics of 562 colistin-resistant isolates. Core genome multi-locus sequence typing (cgMLST) was applied to examine potential transmission events. Results: The presence of ColR-E was observed in all One Health sectors. In Dutch hospitalized patients, ColR-E proportions (11.3 and 11.8% in both measurements) were higher than in Belgian patients (4.4 and 7.9% in both measurements), while the occurrence of ColR-E in Belgian LTCF residents (10.2%) and children in day care centres (17.6%) was higher than in their Dutch counterparts (5.6% and 12.8%, respectively). Colistin use in pig farms was associated with the occurrence of colistin resistance. The percentage of pigs carrying ColR-E was 21.8 and 23.3% in Belgium and 14.6% and 8.9% in the Netherlands during both measurements. The proportion of broilers carrying ColR-E in the Netherlands (5.3 and 1.5%) was higher compared to Belgium (1.5 and 0.7%) in both measurements. mcr-harboring E. coli were detected in 17.4% (31/178) of the screened pigs from 7 Belgian pig farms. Concurrently, four human-related Enterobacter spp. isolates harbored mcr-9.1 and mcr-10 genes. The majority of colistin-resistant isolates (419/473, 88.6% E. coli; 126/166, 75.9% Klebsiella spp.; 50/75, 66.7% Enterobacter spp.) were susceptible to the critically important antibiotics (extended-spectrum cephalosporins, fluoroquinolones, carbapenems and aminoglycosides). Chromosomal colistin resistance mutations have been identified in globally prevalent high-risk clonal lineages, including E. coli ST131 (n = 17) and ST1193 (n = 4). Clonally related isolates were detected in different patients, healthy individuals and livestock animals of the same site suggesting local transmission. Clonal clustering of E. coli ST10 and K. pneumoniae ST45 was identified in different sites from both countries suggesting that these clones have the potential to spread colistin resistance through the human population or were acquired by exposure to a common (food) source. In pig farms, the continuous circulation of related isolates was observed over time. Inter-host transmission between humans and livestock animals was not detected. Conclusions: The findings of this study contribute to a broader understanding of ColR-E prevalence and the possible pathways of transmission, offering insights valuable to both academic research and public health policy development.

This study aimed to map MDRO carriage and potential transmission within and between three Flemish tertiary care hospitals and their neighbouring nursing homes. A cross-sectional MDRO prevalence survey was organized between October 2017 and February 2019. Perianal swabs were cultured for detection of MDRO. Determination of clonal relatedness based on wgMLST allelic profiles was performed. The prevalence of MDRO in Belgian hospitals and NHs is on the rise, compared to previous studies, and transmission in and between institutions is observed. These results re-emphasize the need for a healthcare network-wide infection prevention strategy in which WGS of MDRO strains can be supportive.