ICESp1109, a Novel Hybrid Integrative Conjugative Element of Macrolide-Resistant Streptococcus pyogenes Serotype M77 Collected Between 2003 and 2017 in Poland

Abstract

Background

The antibiotic resistance determinants and associated mobile genetic elements (MGEs) were detected among Streptococcus pyogenes (group A streptococci [GAS]) clinical isolates of an M77 serotype collected in Poland between 2003 and 2017.

Methods

The genomes of 136 M77 GAS isolates were sequenced using short- and selected with long-read approach; whole genome sequences were analyzed to determine the genetic context of macrolide resistance determinants.

Results

The analysed strains were collected from in- and outpatients. Sequencing data analysis revealed that all strains carried the tet(O) gene. They were classified as a single sequence type, ST63. The unique erythromycin-resistance determinant, the erm(TR), was detected in 76.5% (n = 104) of isolates. It was found predominantly (n = 74) within a novel hybrid integrative conjugative element composed of the ICESp1108-like sequence and ICESp2906 variant, which was then named ICESp1109. However, in strains isolated before 2008, erm(TR) was located within ICESp2905 (n = 27) and in 3 strains - within stand-alone ICESp1108-like sequences.

Conclusions

Based on phylogenetic analysis results, the clonal dissemination of the macrolide-resistant S. pyogenes M77/ST63 strain with hybrid ICESp1109 was observed between 2008 and 2017. ICESp1109 is the novel hybrid ICE in gram-positive bacteria.

Streptococcus pyogenes, macrolide resistance, M77/ST63, integrative conjugative element, genome sequencing

Streptococcus pyogenes (group A Streptococcus [GAS]) is a major human pathogen that causes a wide range of infections—from mild, such as pharyngitis or impetigo, to severe and invasive, such as septicemia, necrotizing fasciitis, and streptococcal toxic shock syndrome (for a review, see [1]). Although all GAS strains remain sensitive to β-lactams, and this class of antibiotics is a drug of choice to treat streptococcal infections, macrolides are recommended for patients allergic to β-lactams. Moreover, macrolide combined with β-lactams is preferred in treating severe or complicated S. pyogenes infections [2].

Resistance to macrolides in S. pyogenes primarily involves target site modification and depends on the presence of the erm genes, which code for a methylase of the ribosomal RNA (23S rRNA). The resulting modification of the ribosome prevents the binding of macrolide antibiotics. The erm genes confer resistance not only to macrolides, but also to lincosamides and streptogramin B; therefore, the phenotype presented by the bacteria carrying erm genes is called MLS_B (from macrolides, lincosamides, streptogramin B). The expression of the erm genes is predominantly inducible (iMLS_B); however, the gene can be expressed constitutively (cMLS_B) [3]. In S. pyogenes, few groups of the erm genes have been detected so far—erm(A), with its subtype erm(TR), erm(B), and erm(T). The second resistance mechanism relies on the concomitant action of an efflux pump, which exports macrolide antibiotics out of the bacterial cell, and a protein, which protects the ribosome by driving dissociation of bound macrolide from the ribosome. The associated phenotype, called the M phenotype, is characterized by low-level resistance to 14- and 15-membered macrolides (eg, erythromycin and clarithromycin). This type of resistance in GAS is specified by efflux pumps encoded by mefA or mefE genes, and an msr(D)-encoded ribosome protector [4].

Macrolide resistance genes in GAS are frequently located on integrative and conjugative elements (ICEs). ICEs are diverse mobile genetic structures, distributed in virtually all bacterial genera, integrated into chromosomes, that can excise, circularize, and transfer horizontally via conjugation to neighboring bacteria. Upon integration into bacterial chromosomes, ICEs generate directly repeated sequences (for a review, see [5]). ICEs are important players in bacterial evolution and the lateral spread of antibiotic resistance genes [6, 7].

In the last 2 decades, there has been a gradual increase in the prevalence of macrolide-resistant S. pyogenes (MRSP) isolates in Europe, with some countries rising to over 30%; Italy, Spain, and Greece are among the most affected. Recently, the decline of MRSP isolates has been observed in these countries [8–10]. In multiple studies conducted in different parts of the world, clonal proliferation of certain serotypes of GAS, such as M4, M28, M75, and M77, has been observed [8, 10–12]. One of the main drivers of macrolide resistance in S. pyogenes is the widespread use of macrolide antibiotics for the treatment of respiratory tract infections. The overuse and misuse of antibiotics have led to the emergence and spread of resistant strains, making treatment more difficult and expensive. Continued surveillance and monitoring of MRSP isolates are essential to prevent the spread of resistance and to ensure effective treatment of S. pyogenes infections. In Poland, the level of GAS resistance to erythromycin is estimated in the range of 12%, with a tendency to systematic growth [13]. Recent data estimate the prevalence of resistant strains at around 16%–18% [14]. Although the incidences of S. pyogenes infections are monitored, the data from Central Europe on the strain analysis are missing, so we hope to fill this gap. Here, we present the genomic analysis of MRSP clones of the M77 serotype collected in Poland between 2003 and 2017.

MATERIALS AND METHODS

Streptococcus pyogenes Strains

The 136 S. pyogenes M77 strains were collected between 2003 and 2017 from patients in 2 multicenter surveys, bacterial invasive infections NETwork (BINet) and respiratory tract infections NETwork (Alexander/RESPI-net) [15, 16], concerning community-acquired bacterial infections in Poland: 117 were isolated from respiratory tract infections, 3 from skin infections, 1 from urogenital tract infection, and 15 isolates were invasive (with 12 collected from blood; Supplementary File 1). A general description of these strains is presented in Sitkiewicz et al [14] and included as Supplementary File 1.

Whole Genome Sequencing

Genomic DNA of all isolates was sequenced using a short-read approach with Illumina technology (Illumina Inc). To confirm the structure of mobile genetic elements (MGEs) carrying antibiotic resistance genes, 8 representative strains were selected for the long-read sequencing using Oxford Nanopore technology to obtain complete physical maps. Detailed information regarding the DNA isolation and sequencing strategies are described in Supplementary File 2. Sequence reads obtained during this study were deposited in the Sequence Read Archive database under BioProject ID PRJNA1098028. Complete S. pyogenes M77 genomes were deposited in the National Center for Biotechnology Information GenBank database under accession numbers CP155734–CP155741.

Bioinformatic Analysis

Sequence type (ST) and emm type were determined using SRST2 v.0.2.0 (https://github.com/katholt/srst2). Sequence annotation was performed using DFAST v.1.2.18 (https://github.com/nigyta/dfast_core). Antimicrobial resistance genes and virulence genes were identified using Abricate v.1.0.0 (https://github.com/tseemann/abricate), based on the Comprehensive Antibiotic Resistance Database (CARD) [17] and Virulence Factor Database (VFDB) [18], respectively, retaining only hits with >90% identity and >80% target coverage. Core single-nucleotide polymorphism (SNP) phylogeny was inferred using snippy v.4.6.0 (https://github.com/tseemann/snippy) including masking of the prophage regions, followed by recombination removal by Gubbins [19] and phylogenetic tree construction using FastTree v.2.1 (http://www.microbesonline.org/fasttree/) with a generalized time-reversible model. Pairwise SNPs were calculated using snp-dists v.0.8.2 (https://github.com/tseemann/snp-dists). The data on S. pyogenes M77/ST63 strains isolated in other countries available in public databases were included in the phylogenetic analysis (Supplementary File 3). MGEs were identified within scaffolded genomes using MobileElementFinder v.1.1.2 [20], ICEfinder [21], VRprofile2 [22], and ICEScreen (https://icescreen.migale.inrae.fr/) tools. Prophage sequence detection was conducted using DEPhT v1.2.2 (https://github.com/chg60/DEPhT) and geNomad v.1.7.5 (https://github.com/apcamargo/genomad); manual inspection applying BLASTn [23] was performed to verify the identification results. The phylogenetic tree and heatmap figures presented in this study were visualized in R v.4.3.3 environment using ggtree v.3.10.1 (https://github.com/YuLab-SMU/ggtree) and ComplexHeatmap v.2.18.0 (https://github.com/jokergoo/ComplexHeatmap) packages, respectively.

RESULTS

Strain Characteristics

All 136 GAS with the M77 serotype collected during the 2003–2017 period were tetracycline resistant; the majority of them (104 [76.5%]) were also erythromycin resistant. The macrolide resistance phenotype was assayed according to the European Committee on Antimicrobial Susceptibility Testing (http://www.eucast.org/clinical_breakpoints/); the double disk diffusion test was performed to distinguish between iMLS_B and cMLS_B phenotypes as described by Sitkiewicz et al [14]. Of the erythromycin-resistant strains, 93 exhibited the iMLS_B phenotype, and 11 strains the cMLS_B phenotype [14]. No strains with the M phenotype were detected. The temporal distribution of M77 MRSP collected in Poland over 15 years is presented in Figure 1. The total number of M77 GAS, as well as the MRSP isolates, increased from 2011 till 2016; however, we observed a dramatic drop in the number of isolates in 2017, despite a whole year’s collection of strains. This trend cannot be confirmed, as the M77 GAS strains’ temporal distribution was not analyzed after 2017.

Figure 1.

Temporal distribution of M77/ST63 macrolide-resistant Streptococcus pyogenes isolates in Poland between 2003 and 2017. Abbreviations: GAS, group A streptococci; MRSP, macrolide-resistant Streptococcus pyogenes.

Whole Genome Sequencing Analysis

All 136 strains were analyzed by the short-read whole genome sequencing (WGS) approach, and the 8 representative strains bearing different classes of ICEs carrying antibiotic resistance genes were subjected to long-read sequencing to confirm the correct assembly of those elements. The resulting genomic characteristics are presented in Supplementary File 1. The assembly utilizing long reads is also beneficial in proper genome assembly as the streptococcal chromosomes contain multiple insertion sequences (ISs) [24], which may affect assembly quality when only short reads are used. Genome alignment of S. pyogenes’ complete chromosomes obtained in this study reveals a high degree of synteny and a high level of nucleotide identity to the complete sequence of S. pyogenes NCTC13742 strain (Supplementary Figure 1). The summary of analyzed traits, such as year and anatomical site of isolation, presence of antibiotic resistance genes, and ICE content related to the macrolide resistance is presented in Figure 2.

Figure 2.

Presence/absence matrix depicting the antibiotic resistance (ARG) and virulence gene repertoire detected in Streptococcus pyogenes M77 isolates. The colored squares in the first, second, and third columns from the left represent the year of sample isolation, the strain isolation site, and the type of erm(TR) containing integrative and conjugative elements (ICE), respectively. The shaded squares in ARG heatmap column represent the presence of resistance determinants of macrolides [erm(TR), erm(B), lmrP] and tetracyclines [tet(M) and tet(O)] and the shaded squares in virulence genes column represent a presence/absence matrix of virulence factors.

All of the M77 strains represented ST63. Previously, the M77/ST63 isolates were also reported in Spain [10], Greece [8], the United Kingdom (UK) [25], and Iceland [26] as well as in other European countries [27, 28] and the United States (US), Australia, New Zealand, and Canada [28].

Antibiotic Resistance Genes

The tetracycline determinant detected in all strains was the tet(O) gene (Figure 2), coding for the ribosome protection protein [29]. It is located on an ICE that has 99% nucleotide identity to ICESp2906 described previously for the S. pyogenes strains isolated in Italy [6], except for the 6950-bps segment detected in Polish isolates inserted within orf6 of ICESp2906 (see further description in “tet(O)- and erm(TR)-Containing Elements section”). A single strain, NILSPYO771065, was found to harbor, in addition to tet(O), also tet(M), encoding another type of a ribosome protection protein, located within Tn3872 [30], the Tn916 family member.

All MRSP strains carried erm(TR) as the macrolide resistance determinant, and a single isolate, NILSPYO771065, contained additionally erm(B). The lrmP gene, coding for a proton motive force–dependent drug transporter [31], was detected in 136 genomes, both in macrolide-sensitive and -resistant strains. Therefore, we assume it did not confer macrolide resistance in vivo in the analyzed strains (Figure 2). The mefA, mefE, and msr(D) genes were not identified.

Most erythromycin-resistant strains were isolated between 2008 and 2017 (also considered newer strains in the analysis), and are classified predominantly to clade I based on core SNP analysis (Figure 5). Strains isolated before 2008 are either resistant or sensitive to erythromycin belonging mostly to clade II (older strains) (Figures 2 and  5).

Virulence Genes

Detected GAS virulence factors (VFs) encoded in the core genome and on prophages are summarized in Figure 2. The number of VF genes encoded by a single isolate varies from 14 to 19, with most isolates (∼45%) carrying 17 genes. Thirteen VF genes, including hasABC coding for the capsule, have been identified in all 136 strains. The least frequent VF gene is speA detected in 2 strains. The VF identification results are presented in detail in Supplementary File 4.

Mobile Genetic Elements

Insertion Sequences, Prophages, and Other MGEs

Sequenced M77 GAS strains are similar to other sequenced strains of other GAS serotypes in terms of MGE content. In the analyzed M77 isolates, multiple ISs were identified—IS110, IS21, IS256, IS3, IS30, ISAs1, and ISL3 (Supplementary File 5)—all of which are common in streptococcal genomes [32]. The IS element repertoire identified in scaffolded genomes was validated by analyzing the complete chromosomes of 8 representative strains. The set of ISs detected was consistent across both scaffolded and complete genomes; however, the copy number of 2 ISs varied. An additional ISAs1 copy was found in all complete genomes, while an extra IS3 copy was observed in 2 genomes (Supplementary File 5). These differences were due to the improved resolution provided by the assembly of complete chromosomes utilizing long sequencing reads.

We also identified 2 transposable elements—TnGBS2.3 and Tn3872 [30, 33] (Supplementary File 6). TnGBS2.3 was identified in group B streptococci (GBS), but it was demonstrated to transfer to GAS via conjugation with relatively high frequency. Tn3872, described first in Streptococcus pneumoniae, belongs to the Tn916-family transposons reported initially in Enterococcus faecalis, constituting a large family of ICEs, common in Firmicutes (for a review, see [34]), that can harbor multiple genes conferring antibiotic resistance.

Interestingly, 96 M77 strains carry RD2 elements identified previously in S. pyogenes MGAS6180 and MGAS10270 [35]. This ICE can be transferred via conjugation to multiple GAS serotypes, and to GBS [36]. The RD2 element encodes numerous VFs and is considered the major element affecting the colonization potential of GAS strains [37], as it encodes cell surface–anchored adhesins and R28 protein.

We detected 5 prophages integrated into M77 genomes, namely ϕM77.1 to ϕM77.5 (Figure 3). Their nucleotide sequences are 96%–100% identical to known streptococcal phages. Phages ϕM77.1 and ϕM77.3 may be more common for M77 strains, while phage ϕM77.5 is widely spread in multiple GAS serotypes such as M1 or M12 and M77 (detailed analysis in Supplementary Files 7 and 8).

Figure 3.

Schematic overview of the Polish Streptococcus pyogenes M77/ST63 core chromosome, prophage element insertion sites, and integrative and conjugative elements (ICEs). The circle represents the group A streptococci (GAS) chromosome with the marked nucleotide positions and the origin of replication (ori). The prophage elements are indicated with triangles and ICEs as squares that are shade-coded according to the ICE type; stacked squares indicate ICEs inserted at the same site. Letters denote the integration sites, described in the right panel. The presence of the given element in the sequenced GAS genomes is indicated.

tet(O)- and erm(TR)-Containing Elements

ICESp2905 and Its Variants

The tet(O)-containing element ICESp2906 [6] was detected in all collected M77/ST63 strains. It was either alone, (n = 34; in macrolide-sensitive strains n = 32), or as a hybrid element with IMESp2907 (n = 27), an integrative mobilizable element, constituting ICESp2905 [6] (Figure 4A). ICESp2906 was also detected as a hybrid with an ICESp1108-like element (n = 75) forming a novel element, ICESp1109 (see below). Both ICESp2905 and ICESp1108 [7] are integrated into the M77/ST63 strain genomes between the rum and pnp genes, coding for the 23S rRNA m(5)U(1939)methyltransferase and a phosphorylase superfamily protein [6], respectively. Such integration is catalyzed by a site-specific serine integrase, encoded by a gene located at the 3′ ICE proximity [38].

Figure 4.

Schematic representation of the erm(TR)-containing integrative and conjugative elements (ICEs) identified in the genomes of Streptococcus pyogenes M77/ST63 strains. A, ICESp2905 in the NILSPYO771004 genome and the ICESp2905 variant in the NILSPYO770020 genome. B, The tet(O)- and erm(TR)-containing ICEs in the NILSPYO770002, NILSPYO770111, and NILSPYO771066 genomes. The genomic location of the specific fragment is marked. C, ICESp1109, the novel erm(TR)–tet(O)-containing hybrid element present in the NILSPYO771038 strain. D, Tn3872 of the NILSPYO1065 strain, comprising the tet(M) and erm(B) genes. Tn3872 (GenBank accession number OP715845.1) and Tn916 (GenBank accession number U09422) are shown. The functional modules of Tn916 are marked: conjugation, regulation, recombination, and the accessory gene tet(M). Antibiotic resistance genes as well as the rum and pnp genes are marked as shaded arrows.

In MRSP isolates, IMESp2907 was the source of the erm(TR) gene, similar to what was detected in S. pyogenes in Italy [6, 7]. In 3 strains (NILSPYO770002, NILSPYO770111, and NILSPYO771066), besides ICESp2906 another ICE bearing erm(TR) was detected, with sequence blocks identical in 94%–100% to ICESp1108, and the 2 mentioned ICEs were located in the genome approximately 400 kbps apart from each other (Figure 4B). It should be stressed that compared to the original ICESp2906 sequence [6], the 5′ terminus of ICESp2906 of the 3 mentioned strains contains an insertion of 6950-bp sequence comprising 6 additional orfs. The extended ICESp2906 with such an insertion was identified in all Polish S. pyogenes M77/ST63 strains. Moreover, a structure identical in 99% to the modified ICESp2906 element was also detected in the S. pyogenes NCTC13742 (accession number LS483386.1) and S. pyogenes TSPY453 (accession number CP033337.1) genomes; these are also M77/ST63 strains and were isolated in 2015 in the UK and 2014 in the US, respectively. The origin of the entire 6950-bp fragment is unknown, although fragments identical in 86%–94% to some parts of this sequence (coverage 78%–92%) could be detected in various streptococci—Streptococcus equi subsp zooepidemicus (strains SEZ33, SEZ25, NCTC12090, and others), Streptococcus dysgalactiae subsp equisimilis 89, and Streptococcus suis—but also in the genomes of other bacteria related to the human microbiome such as Filifactor alocis ATCC 35896, or Aerococcus spp.

The ICESp2905 element was identified in 27 isolates collected predominantly in the first years of the analyzed period. Interestingly, the canonical version of this element, identical (99%–100%) to the sequence deposited in the European Molecular Biology Laboratory database (accession number FR691055) [7], was detected in 12 strains while other strains carry the ICESp2905 variants with an insertion within orf6 of this element (Figure 4A). The localization and the composition of individual elements in the representative strains, NILSPYO771004 with ICESp2905 and NILSPYO770020 with the ICESp2905 variant, were confirmed by de novo assembling of their complete chromosomes.

It is worth noticing that in the case of 3 strains, NILSPYO770002 (complete genome assembly), NILSPYO770111 (scaffolded genome), and NILSPYO771066 (scaffolded genome), with distantly located ICESp2906 and the ICESp1108-like elements, ICESp2906 is deprived of its terminal sequences at the 5′ end, and only the terminal 43-bp rum fragment is present; the 3′ end is truncated by the 1055-bp DNA fragment including entire pnp together with terminal 36 bp of the preceding gene (Figure 4B).

ICESp1109, a Novel Hybrid Element

In the majority of strains collected between 2008 and 2017 (n = 75), the ICESp1108-like element (94%–99% nucleotide identity) was inserted in the very 5′ flank of ICESp2906, giving rise to a novel hybrid element that we named ICESp1109 (Figure 4C, Supplementary File 9). Similarly to ICESp2905 and ICESp1108, ICESp1109 integrated between the rum and pnp genes. The genomes of the first collected strains comprising ICESp1109, NILSPYO771038, and NILSPYO771039, and the last one, NILSPYO770049, were assembled into complete chromosomes. So far, the ICESp1109 hybrid element has not been described nor deposited in available public databases. Direct repeats, also known as attachment sites left and right (attL and attR), generated upon ICE integration were detected in NILSPYO771038 for both ICESp1109 components, ICESp1108-like and ICESp2906 variant [7]. The putative sequences, attL and attR of the ICESp1108-like element, were identified within the 3′ end of the rum gene and in the serine recombinase gene, respectively (Supplementary Figure 2). The attL sequence of ICESp2906 was detected to overlap attR of the ICESp1108-like element, and attR of ICESp2906 within the ICESp2906 serine recombinase gene (Supplementary Figure 2). The experimental data are required to determine whether ICESp1109 is active as an entire element.

Tn3872, a tet(M)- and erm(B)-Containing Element

In the genome of the single strain NILSPYO771065, besides the erm(TR) and tet(O) genes detected within ICESp1109, we also identified erm(B) and tet(M), located on Tn3872 (Figure 4D), which is part of a larger element of the Tn5252 superfamily, >62 kb in size (Supplementary Figure 3, detailed analysis in Supplementary File 10).

Phylogenetic Analysis of M77/ST63 Strains

Most M77 strains isolated in Poland and worldwide (>60% according to the PubMLST database (https://pubmlst.org/organisms?title=Streptococcus+pyogenes) are classified as ST63. NILSPYO771001, the first complete genome of the erythromycin-sensitive Polish M77 S. pyogenes strain, was used as a reference genome for the SNP tree construction. The strain was isolated in 2003, that is, earlier than the NCTC13742 type strain, which was collected in 2015. The genomes of the Polish M77/ST63 isolates (n = 136) were compared to M77/ST63 isolates (n = 253) from the UK, Iceland, Spain, Germany, France, Sweden, Canada, Australia, New Zealand, and the US, whose genomes were available in public databases (accessed on 1 March 2024) (Figure 5) [10, 25, 26]. A total of 1344 core genome SNPs were identified in the tested dataset that included strains isolated worldwide. The number of polymorphic sites detected among M77 strains is higher than observed among highly clonal serotypes such as M1 or M3, where differences do not exceed 100 SNPs [39]. The Polish M77 isolates had a maximal pairwise SNP difference of 76 (average difference of 22 core SNPs), and when all M77 isolates were included, the maximal pairwise SNP difference increased to 87 (average difference of 38 core SNPs) (Supplementary File 11).

Figure 5.

The maximum-likelihood core single-nucleotide polymorphism phylogenetic tree of 389 Streptococcus pyogenes M77/ST63 isolates including 136 Polish isolates, and strains from other countries (BioProjects accession numbers indicated in Supplementary File 3). The innermost ring denotes erm(TR) presence or absence; the second ring indicates the relevant integrative and conjugative element presence in the strain; and in the third ring, the year of sample isolation is presented. The clades representing 2 major groups of Polish isolates analyzed in this study are marked in the outermost ring. The NILSPYO771001 genome was used as a reference sequence.

The majority of Polish strains form 2 separated clusters with clonal distribution (clades) (Figure 5). One of them, clade I, groups all of the isolates containing erm(TR) within the ICESp1109 hybrid element (n = 75), NILSPYO771066, NILSPYO770111, and NILSPYO770002—the 3 clones with the distantly inserted ICESp2906 and ICESp1108-like elements in their chromosomes. The first strain (NILSPYO771038), with identified ICESp1109, was isolated in 2008, and in the 2013–2017 period this clone dominated the population of the Polish MRSP strains. In total, 487 core genome SNPs were detected among clade I strains and a maximal pairwise SNP difference of 25 was identified (Supplementary File 11). Clade II contains NILSPYO771001 reference strain and groups the majority of erythromycin-sensitive older clones (with ICESp2906), and 7 of 27 carrying erm(TR) within the ICESp2905 variant element both in older (from 2003 to 2008) and newer (from 2008 to 2017) strains. In total, 235 core genome SNPs were identified in clade II, with a maximal pairwise SNP difference of 20.

The average pairwise difference of 10 core SNPs in clade I and 8 core SNPs in clade II (Supplementary File 11), respectively, indicate their clonal distribution.

Other Polish isolates with ICESp2905 or its variant and a few carrying ICESp2906 cluster predominantly with European isolates (from the UK, Spain, and Iceland) and also with several strains originated from the US, isolated in 2016 and 2017. The majority of the strains isolated in Europe (the UK, Iceland, Spain, Germany) are more diverse than those isolated in the US, which are clonally distributed.

There were no differences between clusters identified by core SNP analysis with strains NILSPYO771001 or NCTC13742 as a reference (Supplementary Figure 4). With S. pyogenes NCTC13742 as a reference, 1339 core genome SNPs were detected among all tested strains. All Polish isolates (n = 136) and a majority of the isolates from other countries (n = 237) carry the tet(O) gene within the ICESp2906 element. No correlation was found between the SNP composition in analyzed strains and their ICE repertoire (Supplementary File 11).

DISCUSSION

The S. pyogenes M77 isolates collected in Poland in 2003–2017 represent a single ST63, which has also been reported in other European countries [8, 10, 25–28], and North America, Australia, and New Zealand [28]. Our phylogenetic analysis of M77/ST63 strains shows relationships between strains isolated in different parts of the globe, suggesting clonal dissemination of analyzed strains. Unfortunately, our observations are limited due to the lack of WGS data from other European countries, especially from Central Europe. In public databases, there were no data from Poland's neighboring countries, such as the Czech Republic, Slovakia, or the eastern part of Europe, and the limited dataset was available from Germany (2 isolates).

The erythromycin-resistant Polish M77/ST63 isolates carry the erm(TR) gene within the tet(O)-containing ICE elements. In the early isolates erm(TR) is found in the already known ICESp2905 [6] or its variant. Starting from 2008 it is located in ICESp1109, the novel hybrid element described in this work, composed of the ICESp2906 variant and ICESp1108-like elements. Although most M77/ST63 strains were analyzed as draft genomic sequences, the genomic organization of MGEs was confirmed by assembling the complete genomes of selected isolates using long-read sequencing. No isolates with erm(TR) without tet(O) were detected, suggesting that ICESp2906 acquisition was a prerequisite for erm(TR) gaining. The strains carrying the ICESp1109 element disseminated clonally in Poland and dominated the population of S. pyogenes M77/ST63. Due to the lack of sequence data from neighboring countries, we cannot determine whether such a clone is disseminated elsewhere.

The surveillance and monitoring of macrolide-resistant S. pyogenes have to be ongoing due to the public health challenges underlined recently also by the World Health Organization in 2024, which added these bacteria to the medium-priority group of pathogens [40].

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Author contributions. J. G. performed the research, analyzed the data, and wrote and revised the manuscript. K. Ż., M. O. S., M. K., I. W.-P., and A. K. performed the research. S. B. B. provided analytical tools. R. G. analyzed the data. J. M. M. provided funding and analytical tools. I. S. conceived the project, provided funding, performed the research, analyzed the data, and wrote the manuscript. I. K.-Z. supervised the project, analyzed the data, and wrote and revised the manuscript.

Ethics compliance. The study was conducted as continuous surveillance following the World Health Medical Association 1966 Declaration of Helsinki and the European Union rules of Good Clinical Practice.

Financial support. The study was funded by the National Science Centre, Poland (grant number 2017/27/B/NZ7/00040); and by the Fondren Foundation. I. S. is a recipient of the Senior Fulbright Award.

Author notes

Present affiliation: Laboratory Diagnostics Department, Międzyleski Specialist Hospital, Warsaw, Poland.

Potential conflicts of interest. The authors: No reported conflicts of interest.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.