antibiotics Communication A New Integrative and Mobilizable Element Is a Major Contributor to Tetracycline Resistance in Streptococcus dysgalactiae subsp. equisimilis Guillem López de Egea 1,2 , Aida González-Díaz 1,2 , Gérard Guédon 3 , Julie Lao 3,4 , Dàmaris Berbel 1,2 , Antonio Casabella 5,6 , José María Marimón 7 , Emilia Cercenado 2,8 , Lucía Fernández-Delgado 1 , Hélène Chiapello 4 , Thomas Lacroix 4 , María Ángeles Domínguez 1,9,10 , Nathalie Leblond-Bourget 3 and Carmen Ardanuy 1,2,9, * 1 Microbiology Department, Hospital Universitari Bellvitge, IDIBELL-UB, 08907 L’Hospitalet de Llobregat, Spain 2 Research Network for Respiratory Diseases (CIBERES), Insituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain 3 DynAMic, Université de Lorraine, Institut national de recherche pour l’agriculture et l’environnement, Faculté des Sciences et Technologies, F-54000 CEDEX Nancy, France 4 MaIAGE, Université Paris-Saclay, Institut national de recherche pour l’agriculture et l’environnement, Domaine de Vilvert, F-78350 CEDEX Jouy-en-Josas, France 5 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 6 Microbiology Department, Hospital Universitari Parc Taulí, 08208 Sabadell, Spain 7 Biodonostia, Infectious Diseases Area, Respiratory Infection and Antimicrobial Resistance Group, Osakidetza Basque Health Service, Donostialdea Integrated Health Organization, Microbiology Department, 20014 San Sebastián, Spain 8 Clinical Microbiology and Infectious Disease Department, Hospital General Universitario Gregorio Marañón, 28009 Madrid, Spain Citation: López de Egea, G.; 9 Department of Pathology and Experimental Therapeutics, School of Medicine and Health Sciences, González-Díaz, A.; Guédon, G.; Lao, University of Barcelona, 08907 Barcelona, Spain 10 Research Network for Infectious Diseases (CIBERINFEC), ISCIII, 28029 Madrid, Spain J.; Berbel, D.; Casabella, A.; Marimón, J.M.; Cercenado, E.; Fernández- * Correspondence: c.ardanuy@bellvitgehospital.cat; Tel.: +34-93-260-7930 Delgado, L.; Chiapello, H.; et al. A New Integrative and Mobilizable Abstract: Tetracycline resistance in streptococci is mainly due to ribosomal protection mediated Element Is a Major Contributor to by the tet(M) gene that is usually located in the integrative and conjugative elements (ICEs) of Tetracycline Resistance in the Tn916-family. In this study, we analyzed the genes involved in tetracycline resistance and the Streptococcus dysgalactiae subsp. associated mobile genetic elements (MGEs) in Streptococcus dysgalactiae subsp. equisimilis (SDSE) equisimilis. Antibiotics 2023, 12, 579. causing invasive disease. SDSE resistant to tetracycline collected from 2012 to 2019 in a single hospital https://doi.org/10.3390/ and from 2018 in three other hospitals were analyzed by whole genome sequencing. Out of a total of antibiotics12030579 84 SDSE isolates, 24 (28.5%) were resistant to tetracycline due to the presence of tet(M) (n = 22), tet(W) Academic Editors: Manuela Oliveira (n = 1), or tet(L) plus tet(W) (n = 1). The tet(M) genes were found in the ICEs of the Tn916-family and Elisabete Silva (n = 10) and in a new integrative and mobilizable element (IME; n = 12). Phylogenetic analysis showed a higher genetic diversity among the strains carrying Tn916 than those having the new IME, Received: 21 February 2023 Revised: 8 March 2023 which were closely related, and all belonged to CC15. In conclusion, tetracycline resistance in SDSE Accepted: 12 March 2023 is mostly due to the tet(M) gene associated with ICEs belonging to the Tn916-family and a new IME. Published: 15 March 2023 This new IME is a major cause of tetracycline resistance in invasive Streptococcus dysgalactiae subsp. equisimilis in our settings. Keywords: Streptococcus dysgalactiae subsp. equisimilis; tet(M); IME; ICE; mobile genetic elements Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Attribution (CC BY) license (https:// Streptococcus dysgalactiae subsp. equisimilis (SDSE) are beta-hemolytic streptococci that creativecommons.org/licenses/by/ possess cell wall carbohydrate serogroup antigens C and G and sometimes A or L. SDSE 4.0/). are present in the microbiota of the respiratory, gastrointestinal, and female genital tract Antibiotics 2023, 12, 579. https://doi.org/10.3390/antibiotics12030579 https://www.mdpi.com/journal/antibiotics Antibiotics 2023, 12, 579 2 of 8 and the skin of humans [1,2]. It usually causes mild to severe infections in humans as Streptococcus pyogenes, ranging from pharyngitis and skin infections to necrotizing fasciitis and streptococcal toxic shock-like syndrome [2,3]. These two species are phylogenetically closely related and share many virulence factors, such as the M-protein, involved in the adhesion and immune system evasion, and used for epidemiological typing [4]. In recent years, two important facts related to SDSE have been described: first, the global emergence of invasive disease due to SDSE [5–8] and second, the description of the horizontal DNA transfer from SDSE to S. pyogenes, such as pbp2X gene, leading S. pyogenes to have a decreased susceptibility to beta-lactams [9]. Tetracycline resistance can be mediated by three main mechanisms: active efflux pumps, ribosomal protection, and enzymatic inactivation. Within the genus Streptococcus, ribosomal protection is the most important mechanism, and the tet(M) gene the most fre- quent. This gene is mostly found in the integrative and conjugative elements (ICEs), mainly of the Tn916-family, which can spread through horizontal transmission by conjugation [10]. In fact, having all the requirements for self-transmission is a hallmark of ICEs. On the other hand, integrative and mobilizable elements (IMEs) are elements that encode their own excision and integration, possess only some sequence and genes involved in conjugation, and hijack the conjugation apparatus of conjugative plasmids or ICEs to promote their own transfer [11]. Despite the fact that IMEs’ prevalence is still underestimated, many of them encode resistance to antibiotics and altogether confer resistance to a large variety of antibiotics [11]. Considering resistance to tetracycline, several IMEs from Streptococcus suis were found to carry the tet(O) gene and one a tet(40) gene [12–14]. However, no IME carrying tet(M) had ever been identified in Firmicutes. During a surveillance study of tetracycline resistance in SDSE we found a new IME carrying the tet(M) gene. Hence, the aim of the present study was to describe this new element and to analyze the epidemiology of tetracycline resistance in SDSE. 2. Results and Discussion Out of a total of 84 SDSE isolates causing invasive disease (iSDSE), 24 (28.5%) were resistant to tetracycline. In order to analyze the trends in the iSDSE over the study period, we analyzed data from the Hospital Universitari de Bellvitge (HUB) covering the whole period. Even though the number of isolates collected per year was low, tetracycline resistance rates remained stable among the iSDSE collected from HUB from 37.5% (3/8) in 2012–2015 to 30.2% (16/53) in 2016–2019. The genome analysis showed that tetracycline resistance was associated with the presence of the tet(M) (n = 22), tet(W) (n = 1), or tet(L) plus tet(W) (n = 1) genes. This gene distribution among the iSDSE agreed with previous results from other regions [15,16]. Nevertheless, exploring the genetic environment of the tet(M) gene, we found that it was carried by two different types of elements transferable by conjugation. In ten isolates, the tet(M) gene was carried by an ICE belonging to the Tn916-family [Tn916 (n = 9) and Tn3872 (n = 1, also having the erm(B) gene)], as previously described. On the other hand, 12 isolates had the tet(M) gene in a new IME (IME_SDSE_HUB-4529; Figures 1 and 2) that was identical (100% nucleotide identity) in all 12 isolates. The IME_SDSE_HUB-4529 encodes a tyrosine integrase and a relaxase of the MOBT superfamily. All of them are integrated in the same site located in an intergenic region between the genes encoding for the alpha subunit of ribonucleotide reductase of class Ib (aerobic) and the Na+ -dependent branched-chain amino acid transporter (Figure 2). A BLASTN search identified identical IMEs (100% identity) in the Enterococcus faecium (Acc. num AP019408.1) and Clostridioides difficile (Acc. num CP016102.1) genomes. The insertion sites of the element in the three species were different suggesting that the integrase is not site-specific. This fact together with the 100% identity of the IMEs and with the clustering of the strains on the phylogenetic tree suggests the clonality of the 12 strains and a recent acquisition of the IME by a common ancestor to these strains. A further BLASTN search identified similar structures in other Gram-positive bacteria (E. faecalis, C. innocuum, and Catenibacterium sp.) commonly found in the gut that the integrase is not site-specific. This fact together with the 100% identity of the IMEs and with the clustering of the strains on the phylogenetic tree suggests the clonality of the Antibiotics 2023, 12, 579 12 strains and a recent acquisition of the IME by a common ancestor to these strains. A3fur-of 8 ther BLASTN search identified similar structures in other Gram-positive bacteria (E. faecalis, C. innocuum, and Catenibacterium sp.) commonly found in the gut microbiota with an iden- tity above 95% (Supplementary Figure S1). The presence of IME structures in iSDSE with a microbiota with an identity above 95% (Supplementary Figure S1). The presence of IME putative origin from other bacterial species is a cause for concern, especially for E. faecium structures in iSDSE with a putative origin from other bacterial species is a cause for concern, which is known to be associated with vancomycin and multidrug resistance. In fact, transfer especially for E. faecium which is known to be associated with vancomycin and multidrug of the van(A) gene from enterococcal species to S. aureus has already been described [17]. resistance. In fact, transfer of the van(A) gene from enterococcal species to S. aureus has The chance of DNA exchanges is more plausible in the current scenario, with an increasing already been described [17]. The chance of DNA exchanges is more plausible in the current frequency of SDSE which is a cause of human infection. scenario, with an increasing frequency of SDSE which is a cause of human infection. Figure Figure 1.1. Phylogenetic Phylogenetic tree tree and and presence/absence presence/absence matrix depicting the acquired resistance resistance genes genes of of iSDSE carrying tet(M). The strain names are shown on the tree branches. The colored squares iSDSE carrying tet(M). The strain names are shown on the tree branches. The colored squares in the in the first column first column represent represent thethe ST ST (Sequence-Type). (Sequence-Type). The The second second column column shows shows the the emm-types. emm-types. The The two two first columns of light blue and grey squares represent the mobile genetic elements (MGEs) first columns of light blue and grey squares represent the mobile genetic elements (MGEs) in which in which the tet(M) gene is found. The following columns represent a presence/absence matrix of tet(M) and the tet(M) gene is found. The following columns represent a presence/absence matrix of tet(M) and other acquired resistance genes indicating in dark blue when present in the same element as tet(M) other acquired resistance genes indicating in dark blue when present in the same element as tet(M) and in dark red when present in a different element. and in dark red when present in a different element. Aside from the elements carrying tetracycline resistance, most of the isolates also had other resistance mechanisms carried by other elements (Figure 1). Among isolates carrying Tn916, one strain had the erm(T) gene in a non-self-transmissible plasmid and the dfr(F) in a different IME; two isolates had the Tn916 inserted into a larger ICE belonging to the Tn1549-family that also had erm(TR); a single isolate had the dfr(F) in a different IME; one isolate had the macrolide efflux genetic assembly (MEGA) element carrying the mef (E) gene; and finally, one isolate also had erm(B) located near an insertion sequence. On the other hand, three out of 12 isolates carrying the IME_SDSE_HUB-4529 also had the erm(TR) gene located in a Tn1549-family of ICE. Table 1 shows the antibiotic susceptibility of the tetracycline-resistant SDSE. Beta- lactams (penicillin and cefotaxime), vancomycin, and linezolid were active against all the strains. In fact, beta-lactams are the first-line treatment of SDSE infections, and linezolid Antibiotics 2023, 12, 579 4 of 8 could be used as an alternative treatment in case of allergy. Regarding levofloxacin, all strains were “susceptible, increased exposure” following the EUCAST criteria. Never- theless, one strain had a D83A substitution in ParC and had a MIC of levofloxacin of Antibiotics 2023, 12, x FOR PEER REVIEW 4 of 2 mg/L which, as has occurred in other streptococci, could be the first step to achieving high-level fluroquinolone resistance under antibiotic pressure. Other resistance rates could be explained by the transferable resistance mechanisms described above. Figure 2. Schematic representation of the IME containing tet(M) in the SDSE compared with the Figure 2. Schematic representation of the IME containing tet(M) in the SDSE compared with the se sequence from E. faecium and C. difficile. This figure shows the structure of the IME containing the quence from E. faecium and C. difficile. This figure shows the structure of the IME containing the tet(M tet(M) gene found in twelve strains of the present study (HUB, HUD, HGUGM) compared with the gene found in twelve strains of the present study (HUB, HUD, HGUGM) compared with the previ previously described isolates in the literature for E. faecium (Acc. num AP019408.1) and C. difficile (Acc. ously described isolates in the literature for E. faecium (Acc. num AP019408.1) and C. difficile (Acc. num num CP016102.1). The size of this IME is 9352 bp. The grey-shaded areas connect identical regions CP016102.1). The size of this IME is 9352 bp. The grey-shaded areas connect identical regions (100% (100% identity). Each arrow represents a gene. Colors represent different genes. Grey: chromosomal identity). Each arrow represents a gene. Colors represent different genes. Grey: chromosomal gene genes (compared in SDSE with reference strain (AC-2713) (Acc num HE858529.1)). Red: integrase; (compared in SDSE with reference strain (AC-2713) (Acc num HE858529.1)). Red: integrase; orange orange: excisionase; yellow: MOBT relaxase; green: tet(M) gene; purple: unknown function genes. excisionase; yellow: MOBT relaxase; green: tet(M) gene; purple: unknown function genes. Table 1. The antibiotic susceptibility of 24 tetracycline-resistant SDSE strains. Aside from the elements carrying tetracycline resistance, most of the isolates also had Antibiotic % Susceptible MIC (50) [mg/L] MIC (90) [mg/L] other resistance mechanisms carried by other elements (Figure 1). Among isolates carrying Penicillin 100 <0.03 <0.03 Tn916, one strain had the erm(T)100 Cefotaxime gene in a non-self-transmissible <0.06 plasmid<0.03and the dfr(F) in a differentErythromycin IME; two isolates had the 54.2Tn916 inserted into <0.25a larger ICE belonging >4 to the Tn1549 Clindamycin 58.3 <0.25 family that also had erm(TR); a single isolate had the dfr(F) in a different IME; one isolate >0.5 Linezolid 100 <1 - had the macrolide Tetracycline efflux genetic 0assembly (MEGA) element - carrying the >4 mef(E) gene; and finally,Chloramphenicol one isolate also had erm(B) 100 located near an insertion 2 sequence. On 2 the other hand Levofloxacin 100 * <1 three out of 12 isolates carrying the IME_SDSE_HUB-4529 also had the erm(TR) gene located <1 Vancomycin 100 <0.5 <0.5 in a Tn1549-family Trimethoprim-of ICE. 91.7 <0.5/9.5 <0.5/9.5 Table 1 shows the antibiotic susceptibility of the tetracycline-resistant SDSE. Beta-lac Sulfamethoxazole tams (penicillin * “Susceptible, and exposure”. increased cefotaxime), vancomycin, and linezolid were active against all the strains. The In fact, beta-lactams are the first-line treatment of SDSE infections, and linezolid phylogenetic analysis of the tetracycline-resistant isolates showed a high STs’ couldandbeemm-type used asdiversity. an alternative treatment We found in case 10 different STsof allergy. (ST15 n = 11,Regarding ST4 n = levofloxacin, 2, ST127 al strains n = were 2, and“susceptible, ST8, ST27, ST31, increased exposure” ST141, DLV8, SLV15,following SLV38 n =the 1), EUCAST criteria. and 13 different Neverthe emm- less,types one (stC839.2 strain had n =a4,D83A substitution stG652.0 in ParC n = 3, stG480.0 n = 2,and hadn a= MIC stG6.1 of levofloxacin 2, stG10.0 of 2 mg/L n = 2, stG245.0 n = 2, and stG2574.3, stG166b.0, stG6746.0, stC36.9, emm131.0, stC5345.1, which, as has occurred in other streptococci, could be the first step to achieving high-leve stG97.0 n = 1) among tetracycline-resistant strains harboring the tet(M). These STs and emm-types have fluroquinolone resistance under antibiotic pressure. Other resistance rates could be ex been previously described as a cause of disease in humans [15,16,18–22]. Figure 1 shows plained by the transferable the phylogenetic resistance relatedness mechanisms of the SDSE described strains carrying tet(M)above. regardless of the MGEs carrying this gene. Of note, the isolates carrying the tet(M) gene in the ICEs of the Tn916 Table 1. The family antibiotic were susceptibility genetically of (Figure more distinct 24 tetracycline-resistant 1) than the strains SDSE strains. carrying tet(M) in the novel IME. In fact, all isolates carrying the IME_SDSE_HUB-4529 belonged to the same clonal Antibiotic % Susceptible MIC (50) [mg/L] MIC (90) [mg/L] Penicillin 100 <0.03 <0.03 Cefotaxime 100 <0.06 <0.03 Erythromycin 54.2 <0.25 >4 Clindamycin 58.3 <0.25 >0.5 Antibiotics 2023, 12, 579 5 of 8 complex (CC15) suggesting a clonal spread of resistance. Nevertheless, these strains of CC15 had different emm-types. In fact, no clear relationship between emm-type and clonal complex has been established for SDSE, as occurs for S. pyogenes, highlighting the more accurate role of CC to differentiate SDSE lineages. Some tetracycline-resistant isolates were also resistant to macrolides, lincosamides, and trimethoprim–sulfamethoxazole. These co-resistances were more frequent in the strains carrying Tn916 (7/10) than in the ones that carry the IME_SDSE_HUB-4529 (3/12) (Figure 1). MGEs are a frequent cause of resistance dissemination in streptococci. Among them, the ICEs had all the genes required for conjugation and could easily spread intra- and inter- species [23]. In this way, we found a high clonal diversity of tetracycline-resistant strains harboring the tet(M) gene in the Tn916. Furthermore, this element has been associated with resistance in streptococci since the late 1990s, enhancing the opportunities to spread among different strains and species [24]. In contrast, we found a high genetic relatedness among isolates having the tet(M) gene in the new IME (ST15), highlighting that the increase in the prevalence of this new element could be associated preferably to clonal spread of the successful lineages having them. Nevertheless, the presence of ICEs harboring other resistance genes in some of the strains suggests a putative scenario of co-dissemination among other streptococci. Furthermore, it is important to note that other ICEs lacking resistance determinants could also be found among strains and could play a role in the spreading of IMEs [11]. In conclusion, the spread of tetracycline resistance in Streptococcus dysgalactiae subsp. equisimilis is mediated by both the horizontal spread of mobile genetic elements among different strains and by the clonal spread of tetracycline-resistant lineages harboring tet(M). We described a new integrative mobilizable element in Streptococcus dysgalactiae subsp. equi- similis with a putative origin from E. faecium or C. difficile, which highlights the importance of interspecies transfer of resistance determinants. 3. Materials and Methods This was a retrospective study from 2012 to 2019 including all tetracycline-resistant iSDSE in the Hospital Universitari de Bellvitge (HUB), an adult tertiary teaching hospital located in the Southern Barcelona area, Spain. Furthermore, in order to understand the spread of MGEs and tetracycline-resistant lineages around the country, isolates causing iSDSE in three other hospitals during 2018 were also included. These additional hospitals were in the Center (H. Universitario Gregorio Marañón, Madrid, HGUGM), North (H. Universitario Donostia, Donostia, HUD), and East (Consorci Sanitari Parc Taulí, Sabadell, CSPT) of Spain. The strains were identified by Matrix-Assisted Laser Desorption/Ionization—Time of Flight (MALDI-TOF, Bruker-Daltonics, Bremen, Germany) and tested by both disc- diffusion and broth microdilution methods for antimicrobial susceptibility following the EUCAST recommendations and criteria (www.eucast.org (accessed on 10 October 2022)). For microdilution, the commercially available plates were used (STRHAE, Sensititre, Ther- moFisher Scientific, Waltham, MA, USA). The following antibiotics were tested: penicillin, ampicillin, cefotaxime, erythromycin, clindamycin, linezolid, tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, vancomycin, and trimethoprim–sulfamethoxazole. The molecular characterization of tetracycline-resistant strains was performed by whole genome sequencing. Genomic DNA was extracted using a QIAamp DNA Mini Kit (Qiagen) and quantified with the QuantiFluor dsDNA System (Promega Corporation, Madi- son, WI, USA). The libraries were prepared using Nextera XT and sequenced by paired-end sequencing (2 × 150 bp) on an Illumina MiSeq Platform (Illumina Inc., San Diego, CA, USA), following the manufacturer’s instructions. The read quality assessment and genome assembly was conducted using the INNUca v4.2 pipeline (github.com/B-UMMI/INNUca (accessed on 10 October 2021)). The multilocus sequence type (MLST) was in silico de- duced using the MLST v2.4 software (github.com/tseemann/mlst (accessed on 10 October 2021)) and the PubMLST database (pubmlst.org/organisms/streptococcus-dysgalactiae Antibiotics 2023, 12, 579 6 of 8 (accessed on 0 October 12021)) and the emm-type by the CDC databases (www2.cdc.gov/ vaccines/biotech/strepblast.asp (accessed on 10 October 2021)). The final assemblies were annotated with Prokka v1.13.7, whereas genes related to conjugative or mobilizable ge- netic elements were annotated with ICEscreen (Lao et al., unpublished results), a tool to detect ICEs and integrative mobilizable elements (IMEs) in Firmicute genomes. The resistance genes were found using the ResFinder database [25]. The genetic environment of the tet(M) gene was studied through comparison with previously described sequences present in public databases using Geneious 9.1.7 (Biomatters, Auckland, New Zealand). For phylogenetic analysis, a single nucleotide polymorphism alignment with Snippy v4.4.0 (github.com/tseemann/snippy (accessed on 11 January 2023)) was performed, the recombi- nation was removed by Gubbins v2.4 [26], and subsequently, a final phylogenetic tree was constructed with RAxML-NG and 100 bootstrap replicates [27], represented using Microre- act (microreact.org/ (accessed on 11 January 2023)). The sequence comparisons between the new IME described in this work and those available on NCBI were displayed using Easyfig program. The sequence data were deposited in the European Nucleotide Archive under the project accession number PRJEB55810 (Supplementary Material Table S1). Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antibiotics12030579/s1, Table S1: Antibiotic resistance and mobile genetic elements in the 24 tetracycline-resistant strains. Figure S1; Schematic representation of the IME containing tet(M) in SDSE (SDSE-HUB-4529) compared with different sequences from E. faecium, C. difficile, Clostridium innocuum, E. faecalis, and Catenibacterium sp. Author Contributions: Conceptualization, C.A., G.L.d.E. and N.L.-B.; methodology, G.L.d.E., A.G.-D., D.B., G.G. and J.L.; software, A.G.-D., J.L., H.C., T.L. and N.L.-B.; validation: G.L.d.E., A.G.-D. and C.A.; formal analysis, A.G.-D., G.G., J.L., N.L.-B., H.C. and T.L.; investigation, G.L.d.E., A.G.-D., D.B., A.C., E.C., J.M.M. and L.F.-D.; resources, M.Á.D. and C.A.; data curation, G.L.d.E., A.G.-D., G.G. and J.L.; writing—original draft, preparation, G.L.d.E., A.G.-D. and C.A.; writing—review and editing, G.L.d.E., A.G.-D., G.G., J.L., D.B., A.C., J.M.M., E.C., L.F.-D., H.C., T.L., M.Á.D., N.L.-B. and C.A.; visualization, D.B. and G.L.d.E.; supervision, C.A. and N.L.-B.; project administration, C.A.; funding acquisition, C.A. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported through the Projects PI21/01000, “Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Respiratorias (CB06/06/0037)” and “CIBER de En- fermededes Infecciosas (CB21/13/0009)”, an initiative of the Instituto de Salud Carlos III, Madrid, Spain. It was co-funded by the European Regional Development Fund/European Social Fund (ERDF/ESF, “Investing in your future”). We thank the CERCA Programme/Generalitat de Catalunya for institutional support. Institutional Review Board Statement: This study was in accordance with the Declaration of Helsinki from the World Medical Association. It was approved by the Clinical Research Ethics Committee of Bellvitge University Hospital (PR283/21). Written informed consent was not required as this was a retrospective and observational study with isolates obtained as part of routine microbio- logical tests. Patient confidentiality was always protected, and all personal data were anonymized following the current legal normative in Spain (LOPD 15/1999 and RD 1720/2007). Moreover, this project followed Law 14/2007 on Biomedical Research for the management of biological samples in clinical research. Informed Consent Statement: Patient consent was waived because acces to electronic clinical records of patients was retrospective, the study involved no intervention, and the results would not be accurate without the collection of all episodes. Data Availability Statement: Sequence data were deposited in the European Nucleotide Archive under the project accession number PRJEB55810. Acknowledgments: We wish to thank the past and present staff of the Microbiology Departments who contributed to this project on a daily basis. This study was partially presented as a poster at the 21st Lancefield International Symposium for Streptococci and Streptococcal Diseases 2022, Stockholm. Conflicts of Interest: The authors declare no conflict of interest. Antibiotics 2023, 12, 579 7 of 8 References 1. Vandamme, P.; Pot, B.; Falsen, E.; Kersters, K.; Devriese, L.A. Taxonomic Study of Lancefield Streptococcal Groups C, G, and L (Streptococcus dysgalactiae) and Proposal of S. dysgalactiae subsp. equisimilis subsp. Nov. Int. J. Syst. Bacteriol. 1996, 46, 774–781. [CrossRef] 2. Efstratiou, A. Pyogenic Streptococci of Lancefield Groups C and G as Pathogens in Man. J. Appl. Microbiol. 1997, 83, 72S–79S. [CrossRef] 3. Brandt, C.M.; Spellerberg, B. Human Infections Due to Streptococcus dysgalactiae subspecies equisimilis. Clin. Infect. Dis. 2009, 49, 766–772. [CrossRef] [PubMed] 4. Jensen, A.; Kilian, M. Delineation of Streptococcus dysgalactiae, Its Subspecies, and Its Clinical and Phylogenetic Relationship to Streptococcus pyogenes. J. Clin. Microbiol. 2012, 50, 113–126. [CrossRef] [PubMed] 5. Lambertsen, L.M.; Ingels, H.; Schønheyder, H.C.; Hoffmann, S.; Hoffmann, S.; Ingels, H.; Lambertsen, L.; Christensen, J.J.; Dessau, R.; Lomborg, S.; et al. Nationwide Laboratory-Based Surveillance of Invasive Beta-Haemolytic Streptococci in Denmark from 2005 to 2011. Clin. Microbiol. Infect. 2014, 20, O216–O223. [CrossRef] [PubMed] 6. Anonymous. Voluntary Surveillance of Pyogenic and Non-Pyogenic Streptococcal Bacteraemia in England, Wales and Northern Ireland: 2020. Health Prot. Rep. 2021, 15, 1–23. 7. Wajima, T.; Morozumi, M.; Hanada, S.; Sunaoshi, K.; Chiba, N.; Iwata, S.; Ubukata, K. Molecular Characterization of Invasive Streptococcus dysgalactiae subsp. equisimilis, Japan. Emerg. Infect. Dis. 2016, 22, 247–254. [CrossRef] [PubMed] 8. Rantala, S.; Vuopio-Varkila, J.; Vuento, R.; Huhtala, H.; Syrjänen, J. Clinical Presentations and Epidemiology of β-Haemolytic Streptococcal Bacteraemia: A Population-Based Study. Clin. Microbiol. Infect. 2009, 15, 286–288. [CrossRef] 9. Beres, S.B.; Zhu, L.; Pruitt, L.; Olsen, R.J.; Faili, A.; Kayal, S.; Musser, J.M. Integrative Reverse Genetic Analysis Identifies Polymorphisms Contributing to Decreased Antimicrobial Agent Susceptibility in Streptococcus pyogenes. mBio 2022, 13, e0361821. [CrossRef] 10. Chopra, I.; Roberts, M. Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiol. Mol. Biol. Rev. 2001, 65, 232–260. [CrossRef] 11. Guédon, G.; Libante, V.; Coluzzi, C.; Payot, S.; Leblond-Bourget, N. The Obscure World of Integrative and Mobilizable Elements, Highly Widespread Elements That Pirate Bacterial Conjugative Systems. Genes 2017, 8, 337. [CrossRef] 12. Libante, V.; Nombre, Y.; Coluzzi, C.; Staub, J.; Guédon, G.; Gottschalk, M.; Teatero, S.; Fittipaldi, N.; Leblond-bourget, N.; Payot, S. Chromosomal Conjugative and Mobilizable Elements in Streptococcus suis: Major Actors in the Spreading of Antimicrobial Resistance and Bacteriocin Synthesis Genes. Pathogens 2019, 9, 22. [CrossRef] 13. Liang, P.; Wang, M.; Gottschalk, M.; Vela, A.I.; Estrada, A.A.; Wang, J.; Du, P.; Luo, M.; Zheng, H.; Wu, Z. Genomic and Pathogenic Investigations of Streptococcus suis Serotype 7 Population Derived from a Human Patient and Pigs. Emerg. Microbes. Infect. 2021, 10, 1960–1974. [CrossRef] 14. Wang, J.; Qi, K.; Bai, X.; Wu, Z.; Kang, W.; Liang, P.; Zheng, H.; Xu, J. Characterization of Integrative and Conjugative Elements Carrying Antibiotic Resistance Genes of Streptococcus suis Isolated in China. Front. Microbiol. 2022, 13, 1074844. 15. Lu, B.; Fang, Y.; Huang, L.; Diao, B.; Du, X.; Kan, B.; Cui, Y.; Zhu, F.; Li, D.; Wang, D. Molecular Characterization and Antibiotic Resistance of Clinical Streptococcus dysgalactiae subsp. equisimilis in Beijing, China. Infect. Genet. Evol. 2016, 40, 119–125. [CrossRef] 16. Rojo-Bezares, B.; Toca, L.; Manuel Azcona-Gutiérrez, J.; Ortega-Unanue, N.; Toledano, P.; Sáenz, Y. Streptococcus dysgalactiae subsp. equisimilis from Invasive and Non-Invasive Infections in Spain: Combining Epidemiology, Molecular Characterization, and Genetic Diversity. Eur. J. Clin. Microbiol. Infect. Dis. 2021, 40, 1013–1021. 17. Mcguinness, W.A.; Malachowa, N.; Deleo, F.R. Vancomycin Resistance in Staphylococcus aureus. Yale J. Biol. Med. 2017, 90, 269–281. 18. Gherardi, G.; Imperi, M.; Palmieri, C.; Magi, G.; Facinelli, B.; Baldassarri, L.; Pataracchia, M.; Creti, R. Genetic Diversity and Virulence Properties of Streptococcus dysgalactiae subsp. equisimilis from Different Sources. J. Med. Microbiol. 2013, 63, 90–98. 19. de Souza, J.P.; Santos, A.R.; de Paula, G.R.; Barros, R.R. Antimicrobial Susceptibility and Genetic Relationships among Streptococcus dysgalactiae subsp. equisimilis Isolates in Rio de Janeiro. Infect. Dis. 2016, 48, 676–681. [CrossRef] 20. Leitner, E.; Zollner-Schwetz, I.; Zarfel, G.; Masoud-Landgraf, L.; Gehrer, M.; Wagner-Eibel, U.; Grisold, A.J.; Feierl, G. Prevalence of Emm Types and Antimicrobial Susceptibility of Streptococcus dysgalactiae subsp. equisimilis in Austria. Int. J. Med. Microbiol. 2015, 305, 918–924. [CrossRef] 21. Traverso, F.; Blanco, A.; Villalón, P.; Beratz, N.; Sáez Nieto, J.A.; Lopardo, H. Molecular Characterization of Invasive Streptococcus dysgalactiae subsp. equisimilis. Multicenter Study: Argentina 2011–2012. Rev. Argent. Microbiol. 2016, 48, 279–289. [CrossRef] 22. Loubinoux, J.; Plainvert, C.; Collobert, G.; Touak, G.; Bouvet, A.; Poyart, C. Adult Invasive and Noninvasive Infections Due to Streptococcus dysgalactiae subsp. equisimilis in France from 2006 to 2010. J. Clin. Microbiol. 2013, 51, 2724–2727. [CrossRef] 23. Ambroset, C.; Coluzzi, C.; Guédon, G.; Devignes, M.D.; Loux, V.; Lacroix, T.; Payot, S.; Leblond-Bourget, N. New Insights into the Classification and Integration Specificity of Streptococcus Integrative Conjugative Elements through Extensive Genome Exploration. Front. Microbiol. 2016, 6, 1483. [CrossRef] [PubMed] 24. Rice, L.B. Tn916 Family Conjugative Transposons and Dissemination of Antimicrobial Resistance Determinants. Antimicrob. Agents Chemother. 1998, 42, 1871–1877. [CrossRef] [PubMed] 25. Zankari, E.; Hasman, H.; Cosentino, S.; Vestergaard, M.; Rasmussen, S.; Lund, O.; Aarestrup, F.M.; Larsen, M.V. Identification of Acquired Antimicrobial Resistance Genes. J. Antimicrob. Chemother. 2012, 67, 2640–2644. [CrossRef] [PubMed] Antibiotics 2023, 12, 579 8 of 8 26. Croucher, N.J.; Page, A.J.; Connor, T.R.; Delaney, A.J.; Keane, J.A.; Bentley, S.D.; Parkhill, J.; Harris, S.R. Rapid Phylogenetic Analysis of Large Samples of Recombinant Bacterial Whole Genome Sequences Using Gubbins. Nucleic Acids Res. 2015, 43, e15. [CrossRef] 27. Kozlov, A.M.; Darriba, D.; Flouri, T.; Morel, B.; Stamatakis, A. RAxML-NG: A Fast, Scalable and User-friendly Tool for Maximum Likelihood Phylogenetic Inference. Bioinformatics 2019, 35, 4453–4455. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.