Enterococcus faecium

We used PanACoTA⁴⁸ version 1.2.0 to build phylogenies for 15 bacterial species (Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes, Salmonella enterica, Listeria monocytogenes, Helicobacter pylori, Mycobacterium tuberculosis, Neisseria meningitidis, Staphylococcus aureus, Bacillus subtilis, Campylobacter jejuni, Klebsiella pneumoniae, Bacillus velezensis, Acinetobacter baumannii, Enterococcus faecium). PanACoTA allows phylogenetic tree reconstructions based on the core genomes. For each of the species, we took all genomes under a nucleic acid format in NCBI (fna) and annotated them using prodigal (PanACoTA annotate options–cutn 10000–l90 400 –prodigal). We then computed the pangenome and coregenome (PanACoTA pangenome; PanACoTA corepers; with default parameters). Finally, we aligned the coregenome (PanACoTA align, default parameters) and computed a phylogenetic tree (PanACoTA tree, -b 1000). For this step PanACoTA, uses IQTree^{49 (link)}, (version 2.1.4) and the following option (iqtree -m GTR -bb1000 -st DNA).

Details of Illumina read sets used in this study are provided in Table 1 and Table 2. Data tables specifying the expected STs of each read set, summarised from published papers, are given in Additional file 1 [35 ].Table 1

Data sets used to assess accuracy of SRST2

Species	Citation	N (isolates)	Population	Sequencing centre	Average read depth	Read length (bp)
Staphylococcus aureus	[36 (link)]	134	Clonal, ST22	Sanger, UK	24×	55
Staphylococcus aureus	[37 ]	128	Clonal, ST239	Sanger, UK	60×	65
Streptococcus pneumoniae	[38 (link)]	113	Clonal, ST81	Sanger, UK	30×	55
Salmonella enterica Typhimurium	[39 (link)]	44	Clonal, ST313	Sanger, UK	34×	76
Shigella (E. coli)	[40 (link)]	81	Clonal, S. sonnei	Sanger, UK	25×	55
Enterococcus faecium	[41 (link)]	43	Diverse, dominated by ST203, ST17	Melbourne, Australia	658×	101
Listeria monocytogenes	This paper	231	Diverse	Melbourne, Australia	36×	152

Table 2

Data sets used to demonstrate utility of SRST2 in the hospital setting

Species	Citation	N (isolates)	Average read depth	Read length (bp)
Enterococcus faecium (Figure 7a-c)	[41 (link)]	43	658×	101
Hospital outbreak investigations (Figure 8a-b)	[15 (link)]	20	36×	151
K. pneumoniae, E. coli	[42 (link)]	69, 74	34×	101

EU MSs reported mandatory data collected following AMR monitoring programs during 2020 and 2021. ‘Directive 2003/99/EC requires Member States to ensure that monitoring provides comparable data on the occurrence of antimicrobial resistance (‘AMR’) in zoonotic agents and, in so far as they present a threat to public health, other agents’. ‘Directive 2003/99/EC also requires Member States to assess the trends and sources of AMR in their territory and to transmit a report every year covering data collected in accordance with that Directive to the Commission.’ Furthermore, some non‐EU countries reported AMR data and both, some EU and non‐EU reporting countries (RCs) also reported voluntary data from samples that were not included in the mandatory programs per reporting year.
The Commission Implementing Decision 2013/652/EU³⁰ lays down detailed rules for the harmonised monitoring and reporting of AMR in zoonotic and commensal bacteria applicable until 31 December 2020. The Commission Implementing Decision (EU) 2020/1729³¹ of 17 November 2020 lays down new rules for antimicrobial resistance monitoring performed in 2021 onwards. This Decision specifies harmonised rules for the period 2021–2027 for the monitoring and reporting of AMR to be carried out by Member States in accordance with EU Regulations.
The Commission Implementing Decision (EU) 2020/1729 determines specific technical requirements for AMR testing and reporting in relation to sampling in food‐producing animals and derived meat (at retail and at border control posts). The Commission Implementing Decision (EU) 2020/1729 indicates that the monitoring and reporting of AMR shall cover the following bacteria: (a) Salmonella spp.; (b) Campylobacter coli (C. coli); (c) Campylobacter jejuni (C. jejuni); (d) Indicator commensal Escherichia coli (E. coli); (e) Salmonella spp. and E. coli producing the following enzymes: (i) Extended Spectrum β‐Lactamases (ESBL); (ii) AmpC β‐Lactamases (AmpC); (iii) Carbapenemases (CP). Therefore, during 2021, AMR data were collected from the bacteria listed above. It seems relevant to notice that the collection of AMR data from Campylobacter coli isolates was not compulsory in 2020. Despite this, some countries reported AMR data related to Campylobacter coli.
Countries can also report AMR data from other agents of public health importance such as methicillin‐resistant Staphylococcus aureus (MRSA). According to Commission Implementing Decision(EU) 2020/1729 the monitoring and reporting of AMR may also cover indicator commensal Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium).
A scientific report published by EFSA in 2012 included technical specifications on the harmonised monitoring and reporting of antimicrobial resistance in methicillin‐resistant Staphylococcus aureus (MRSA) in food‐producing animals and food (EFSA, 2012 (link)). Detailed rules were specified for harmonised monitoring and reporting on the prevalence of resistant microorganisms in food‐producing animals and food, in particular as regards the microorganisms to be included, the origin of the isolates of the microorganisms, the number of isolates to be tested, the antimicrobial susceptibility tests to be used, the specific monitoring of MRSA and ESBL‐ or AmpC‐producing bacteria and the collection and reporting of the data. Comparison between human data and data from food‐producing animals and food sector was ensured by involvement of ECDC.
The Commission Implementing Decision (EU) 2020/1729 specifies that the monitoring and reporting of AMR shall cover the following food‐producing animal populations and food: (a) broilers; (b) laying hens; (c) fattening turkeys; (d) bovine animals under 1 year of age; (e) fattening pigs; (f) fresh meat from broilers; (g) fresh meat from turkeys; (h) fresh meat from pigs; (i) fresh meat from bovine animals. This European Commission Decision indicates the sampling frequency for MSs to carry out the AMR monitoring and reporting in accordance with the following rotational system: (a) In the years 2021, 2023, 2025 and 2027: in fattening pigs, bovine animals under 1 year of age, pig meat and bovine meat. (b) In the years 2022, 2024 and 2026: in laying hens, broilers, fattening turkeys and fresh meat derived from broilers and turkeys.
Therefore, following relevant EU legislation AMR data presented in this Report were collected from poultry populations and derived meat thereof in 2020 and from pigs and from bovines under 1 year of age in 2021.
The Commission Implementing Decision (EU) 2013/652 and the Commission Implementing Decision(EU) 2020/1729 lay down detailed rules for sampling design and sample size as well as for antimicrobial susceptibility testing for the different bacteria. These European Commission Decisions indicate the analytical methods for detection and antimicrobial susceptibility testing that shall be performed by the laboratories referred to in Article 3(2). AMR testing shall be performed by using the broth microdilution method according to the reference method ISO 20776‐1:2019.
For AMR testing, isolates were obtained through harmonised national programs. The broth microdilution testing method was widely used for susceptibility testing following EU legislation.
On November 17, 2020, the European Commission laid down the new technical specifications in Commission Implementing Decision (EU) 2020/1729 and repealed Commission Implementing Decision(EU) 2013/652. The new legislation came into effect on 1 January 2021, and updates technical specifications for harmonised AMR monitoring and reporting to include the monitoring of AMR in derived meat sampled at border control posts, the testing of new substances. The new legislation also authorises WGS as an alternate method to phenotypic testing for AMR monitoring. The new rules apply until December 2027.
Resulting quantitative³² isolate‐based data were reported to EFSA and considered for this report. Resistance was interpreted using EUCAST ECOFF values (see text box below for further information). The antimicrobials incorporated in this report were selected based on their public health relevance and as representatives of different antimicrobial classes. Data on methicillin resistant Staphylococcus aureus (MRSA) and other microorganisms apart from those required by legislation were reported on a voluntary basis.

Whole cell ELISA was used to determine the specificity of mAbs 9H8 and 10H8 across different clinically important pathogens. The production of 9H8 and 10H8 mAbs has been described previously (He et al., 2022 (link)).
Candida albicans cells were adjusted to an OD600 of 0.5 in a 0.05 M carbonate buffer solution (CBS) that contained 15 mM Na₂CO₃ and 35 mM NaHCO₃ at pH9.6. The prepared suspensions were then used to coat the ELISA plates (100 μl/well) overnight at 4°C. As the negative controls, a panel of clinically relevant organisms including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermis, Cryptococcus neoformans, C. tropicalis, N. glabrata, C. parapsilosis, C. krusei (P. kudriavzevii), Saccharomyces cerevisiae, and Candida dublinensis were used. These strains were grown, resuspended in CBS (OD600 = 0.5) and then coated onto ELISA plates. The plates were washed three times with PBS containing 0.05% Tween 20 (PBST), blocked with 100 μl of PBST plus 3% (w/v) bovine serum albumin (BSA), and incubated for 2 h at 37°C. The plates were treated for 1 h at 37°C with 10H8 or 9H8 that were diluted to 1 μg/ml by PBST plus 1% (w/v) BSA, followed by addition of 1:5,000 diluted horseradish peroxidase conjugated (HRP) goat anti-mouse IgG (Solarbio, Beijing China). After washing, 100 μl of the 3,3′,5,5′-tetramethylbenzedine (TMB) solution (Beyotime, Shanghai, China) was added to each well and incubated at 37°C for 10 min. The reaction was stopped with 50 μl of 2 N H₂SO₄, and the OD450 values were finally measured with the VersaMax plate reader (Molecular Devices, CA, United States).