Epidemiology and genetic diversity of linezolid resistant enterococci clinical isolates in Belgium from 2013 to 2021.

OBJECTIVES: Linezolid resistant opportunistic human pathogens Enterococcus faecalis and Enterococcus faecium are an emerging health threat as limited therapeutic options remain. The aim was to investigate the epidemiology, resistance mechanisms and genetic diversity of Belgian linezolid resistant enterococci (LRE) isolated between 2013 and 2021 received at the Belgian National Reference Centre (NRC) for Enterococci. METHODS: Linezolid susceptibility testing was performed upon request on 2458 submitted Enterococci strains. Whole genome sequencing was performed on all LRE strains. RESULTS: Seventy-eight LRE human isolates, of which 63 (81%) E. faecalis and 15 (19%) E. faecium strains, were submitted to the Belgian NRC for Enterococci. Of the linezolid resistant E. faecalis strains, 97% harboured the optrA (56% wild-type pE349) and 3% the poxtA gene. Of the linezolid resistant E. faecium strains, 54% harboured the G2576T point mutation in the V domain of the 23S rRNA genes, 23% the poxtA and 23% the optrA gene. Furthermore two E. faecium strains were identified with a combination of two resistance mechanisms ((i) optrA and poxtA and (ii) cfr(B) and G2576T point mutation respectively). Vancomycin resistance was observed in 15% (n=12) of the LRE. ST480 (n=42/63 typed strains, 67%) was the most frequently detected sequence type (STs) in linezolid resistant E. faecalis strains, while ST203 (n=5/15 typed strains, 33%) was the most frequently detected STs in linezolid resistant E. faecium strains. CONCLUSIONS: E. faecalis isolates harbouring optrA were the predominant LRE in Belgium with ST480 as the most prominent MLST. Linezolid resistance in E. faecium could be attributed to either chromosomal mutations or transferable resistance determinants.

One Health surveillance of colistin-resistant Enterobacterales in Belgium and the Netherlands between 2017 and 2019.

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.

Clinical and molecular epidemiological features of critically ill patients with invasive group A Streptococcus infections: a Belgian multicenter case-series.

BACKGROUND: Recent alerts have highlighted an increase in group A streptococcal (GAS) infections since 2022 in Europe and the United States. Streptococcus pyogenes can cause limited skin or mucosal disease, but can also present as severe invasive disease necessitating critical care. We performed a multicenter retrospective study of patients with GAS infections recently admitted to Belgian intensive care units (ICUs) since January 2022. We describe patient characteristics and investigate the molecular epidemiology of the S. pyogenes strains involved. RESULTS: Between January 2022 and May 2023, a total of 86 cases (56 adults, 30 children) with GAS disease were admitted to critical care in the university hospitals of Leuven, Antwerp and Liège. We noted a strikingly high incidence of severe community-acquired pneumonia (sCAP) (45% of adults, 77% of children) complicated with empyema in 45% and 83% of adult and pediatric cases, respectively. Two-thirds of patients with S. pyogenes pneumonia had viral co-infection, with influenza (13 adults, 5 children) predominating. Other disease presentations included necrotizing fasciitis (23% of adults), other severe skin/soft tissue infections (16% of adults, 13% of children) and ear/nose/throat infections (13% of adults, 13% of children). Cardiogenic shock was frequent (36% of adults, 20% of children). Fifty-six patients (65%) had toxic shock syndrome. Organ support requirements were high and included invasive mechanical ventilation (77% of adults, 50% of children), renal replacement therapy (29% of adults, 3% of children) and extracorporeal membrane oxygenation (20% of adults, 7% of children). Mortality was 21% in adults and 3% in children. Genomic analysis of S. pyogenes strains from 55 out of 86 patients showed a predominance of emm1 strains (73%), with a replacement of the M1global lineage by the toxigenic M1UK lineage (83% of emm1 strains were M1UK). CONCLUSIONS: The recent rise of severe GAS infections (2022-23) is associated with introduction of the M1UK lineage in Belgium, but other factors may be at play-including intense circulation of respiratory viruses and potentially an immune debt after the COVID pandemic. Importantly, critical care physicians should include S. pyogenes as causative pathogen in the differential diagnosis of sCAP.

Epidemiology and molecular typing of multidrug-resistant bacteria in tertiary hospitals and nursing homes in Flanders, Belgium.

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.

Increase in bloodstream infections caused by emm1 group A Streptococcus correlates with emergence of toxigenic M1UK, Belgium, May 2022 to August 2023.

Many European countries have recently reported upsurges in invasive group A Streptococcus (iGAS) infections, mainly caused by emm1 Streptococcus pyogenes, specifically the toxigenic M1UK lineage. We present the epidemiology of emm1 causing iGAS in Belgium during 2018-August 2023, and describe an emergence of the toxigenic M1UK lineage in Belgium in mid-2022 that was observed as an increase in bloodstream infections caused by emm1 S. pyogenes that continued into 2023.

Epidemiology and molecular typing of multidrug-resistant bacteria in day care centres in Flanders, Belgium.

The global prevalence and spread of multidrug-resistant organisms (MDROs) represent an emerging public health threat. Day care centre (DCC) attendance is a risk factor for MDRO carriage in children and their environment. This study aimed to map the epidemiology of carriage and potential transmission of these organisms within 18 Flemish DDCs (Belgium). An MDRO prevalence survey was organised between November 2018 and February 2019 among children attending the centres. Selective chromogenic culture media were used for the detection of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E), carbapenemase-producing Enterobacterales (CPE), and vancomycin-resistant Enterococci (VRE) in faecal swabs obtained from diapers or jars (n = 448). All isolated MDROs were subjected to resistance gene sequencing. A total of 71 of 448 samples (15.8%) yielded isolates of ESBL-E with a predominance of Escherichia coli (92.2% of ESBL-E) and ESBL resistance gene blaCTX-M-15 (50.7% of ESBL coding genes in E. coli). ESBL-E prevalence varied between DCCs, ranging from 0 to 50%. Transmission, based on the clonal relatedness of ESBL-E strains, was observed. CPE was identified in only one child carrying an E. coli with an OXA-244 gene. VRE was absent from all samples. The observed prevalence of ESBL-E in Flemish DCCs is high compared with previous studies, and our findings re-emphasise the need for rigorous hygiene measures within such centres to control the further spread of MDROs in the community.

Implementation of the Infection Risk Scan (IRIS) in nine hospitals in the Belgian-Dutch border region (i-4-1-Health project).

BACKGROUND: A tool, the Infection Risk Scan has been developed to measure the quality of infection control and antimicrobial use. This tool measures various patient-, ward- and care-related variables in a standardized way. We describe the implementation of this tool in nine hospitals in the Dutch/Belgian border area and the obtained results. METHODS: The IRIS consists of a set of objective and reproducible measurements: patient comorbidities, (appropriate) use of indwelling medical devices, (appropriate) use of antimicrobial therapy, rectal carriage of Extended-spectrum beta-lactamase producing Enterobacterales and their clonal relatedness, environmental contamination, hand hygiene performance, personal hygiene of health care workers and presence of infection prevention preconditions. The Infection Risk Scan was implemented by an expert team. In each setting, local infection control practitioners were trained to achieve a standardized implementation of the tool and an unambiguous assessment of data. RESULTS: The IRIS was implemented in 34 wards in six Dutch and three Belgian hospitals. The tool provided ward specific results and revealed differences between wards and countries. There were significant differences in the prevalence of ESBL-E carriage between countries (Belgium: 15% versus The Netherlands: 9.6%), environmental contamination (median adenosine triphosphate (ATP) level Belgium: 431 versus median ATP level The Netherlands: 793) and calculated hand hygiene actions based on alcohol based handrub consumption (Belgium: 12.5/day versus The Netherlands: 6.3/day) were found. CONCLUSION: The Infection risk Scan was successfully implemented in multiple hospitals in a large cross-border project and provided data that made the quality of infection control and antimicrobial use more transparent. The observed differences provide potential targets for improvement of the quality of care.

Harmonization of whole-genome sequencing for outbreak surveillance of Enterobacteriaceae and Enterococci.

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.