Evidence of transfer of antimicrobial resistance genes from the porcine pathogen Streptococcus suis to human clinical isolates of Streptococcus agalactiae in a major pig-producing region of Spain

Abstract

The emergence of antimicrobial resistance (AMR) is a global threat, with livestock antibiotic use proposed as a significant contributor. We investigated Streptococcus suis, a multidrug-resistant porcine pathogen, as a potential source of AMR gene dissemination to human-associated streptococci in Aragón, Spain. Among 91 porcine isolates recovered across Spain, erythromycin and tetracycline resistance was linked to erm(B) and tet(O) genes, frequently co-located on Tn5252-family integrative and conjugative elements (ICEs) and integrative and mobilizable elements (IMEs). Tn5252-family ICEs shared 32–100% of their sequence and carried different AMR gene combinations. Screening of 2388 human clinical streptococcal isolates of different species obtained in Aragón revealed ∼20% erythromycin resistance, particularly S. pneumoniae and S. agalactiae. Analysis of erm(B)-tet(O) linkage in a set of erythromycin-resistant isolates and subsequent genome sequencing, revealed twelve isolates carrying Tn5252-family ICEs with both AMR genes. Eight S. agalactiae isolates harbored Tn5252-family ICEs with >95% nucleotide identity across >91% of their sequence compared with two different ICEs identified in S. suis isolates recovered in the same region and period. Conjugation assays confirmed ICE transfer from S. suis to S. agalactiae, S. pneumoniae, and S. pyogenes, while in vitro experiments demonstrated that recombination between ICEs promotes diversification of AMR gene cargo within ICEs. Our results identify S. suis as a reservoir and generator of ICEs carrying distinct AMR gene combinations that can be horizontally transferred to other human pathogenic streptococci, leading to multidrug resistance in a single step. These findings provide strong evidence supporting interspecies AMR dissemination in regions with intensive pig production and underscore the need for integrated One Health strategies combining veterinary and clinical genomic surveillance, and targeted interventions to limit the spread of mobile genetic elements across species.

Graphical abstract

1. Introduction

The emergence and spread of antimicrobial resistance (AMR) are major global health challenges. Extensive antibiotic use in animal production may contribute to AMR dissemination by selecting resistant zoonotic bacteria and facilitating gene transfer to human pathogens [1], [2]. Therefore, identifying the bacterial species and mechanisms involved in this transfer is essential for designing effective strategies limiting the spread of AMR from veterinary to human settings.

Streptococcus suis is a Gram-positive commensal of porcine upper respiratory tract. Under certain conditions, it can cause sepsis and meningitis [3]. Up to 100% of pigs can harbour S. suis on a farm, and over 60–80% of European pig farms are affected [4]. Due to the lack of effective vaccines, antibiotics remain the primary treatment, representing 5–20% of antibiotic use in pig farming [4]. As a commensal, S. suis is exposed to antibiotics targeting other pathogens, which has driven high AMR rates globally [5], [6]. S. suis can also colonize humans, with carriage rates up to 20% in high-risk populations [7]. Moreover, it is an emerging zoonotic pathogen causing sepsis and meningitis, with recent European incidence estimates ranging from 0.1 to 4.9 cases per 100.000 persons in the at-risk population [8].

S. suis harbors diverse mobile genetic elements (MGEs), including prophages, plasmids, transposons, integrative conjugative elements (ICEs), and integrative and mobilizable elements (IMEs), which facilitate the acquisition and dissemination of AMR genes [5], [9]. Comparative analyses have suggested horizontal transfer of AMR genes between S. suis and other bacteria [6], [10]. However, most evidence has been derived from in vitro experiments and genomic comparisons, leaving uncertainty about whether such gene transfers occur in real-world epidemiological settings. Considering its high prevalence in pigs, the widespread of AMR, and its capacity to colonize humans and other species, this study aimed to investigate the role of S. suis in the emergence of AMR in human-pathogenic streptococci and to elucidate underlying mechanisms. We hypothesized that if this process occurs, it is most likely to take place in regions with intensive pig production. Spain is the largest pig producer in Europe, with Aragón as its leading pig-producing region. Therefore, it was chosen as the focus of our study.

2. Material and methods

2.1. Bacterial and growth conditions

The isolates used and produced in this study are listed in Table S1 and summarized in Text S1. The collection includes 91 S. suis clinical isolates previously described obtained from 11 autonomous communities in Spain (2014–2020), representing 10 serotypes and 36 Sequence Types (ST) by multi locus sequence typing (MLST) [11], [12]. The S. suis reference strain P1/7 [13] and its fluorescent spectinomycin-resistant mutant derivative P1/7∆gfp+ [14] were also included. In addition, 2423 clinical isolates from S. pneumoniae (n = 539), S. agalactiae (n = 1519), and S. pyogenes (n = 365) recovered from human patients at Miguel Servet University-Hospital (Zaragoza, Spain) between 2019 and 2021, were included. These strains were identified using MALDI-TOF MS and API Strep (BioMérieux), as part of routine diagnostic procedures. As part of this study, several mutants were generated, and they are described in next sections. Specifically, these included 4 spontaneous rifampicin resistant mutants, Ss_45r, Ss_124r, SagS1R1, SpyS1R1, derived from Ss_45, Ss_124, SagS1R1, SpyS1R1, respectively, a P1/7 transconjugant P1/7ICE115, derived from P1/7, and its derivative mutant P1/7ICE115∆tetO, and 6 transformants derived from Ss_45 and Ss_124 (Ss_45r_Tf25, Ss_45r_Tf29, Ss_45r_Tf33, Ss_124r_Tf5, Ss_124r_Tf8, and Ss_124r_Tf10). S. suis isolates were grown in Todd-Hewitt Broth (THB, Oxoid) with 15% Agar (THA), while for the remaining streptococcal species, 5% Sheep Blood was added to the medium. All strains were incubated in a candle jar at 37 °C for 24 h. For bacterial liquid cultures, the bacteria were propagated in THB, starting with an Optical Density at 600 nm (OD₆₀₀) of 0.05 as described [11].

2.2. Antibiotic resistance determination

Antimicrobial susceptibility to penicillin, clindamycin, erythromycin and tetracycline was evaluated on all clinical strains obtained from human patients and several derivative mutants according to EUCAST recommendations [15], using disk diffusion (Oxoid®) and the microdilution method with the automated Microscan WalkAway (Beckman Coulter) system that enables the determination of the minimal inhibitory concentration.

2.3. Whole genome sequencing and bioinformatics

Strains resistant to tetracycline and erythromycin and carrying the genes tet(O) and erm(B) in S. suis and S. agalactiae were selected for genome sequencing. For S. suis, the size of the PCR amplicon spanning the tet(O) and erm(B) loci was used as an additional selection criterion, with the aim of capturing a diverse set of MGEs. Together, 11 S. suis isolates (Ss_27, Ss_31, Ss_50, Ss_61, Ss_64, Ss_81, Ss_105, Ss_110, Ss_146, Ss_160, and Ss_165) and 11 S. agalactiae isolates (Sa_26, Sa_37, Sa_44, Sa_48, Sa_56, Sa_75, Sa_79, Sa_82, Sa_83, Sa_85, and Sa_86) were selected, and their chromosomal DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, USA). DNA-Seq library preparation and sequencing were performed at STAB Vida Lda (Caparica, Portugal) using Illumina. Chromosomal DNA from the transformants was extracted with same kit but sequenced at Plasmidsaurus Inc. (Louisville, USA) using nanopore. Raw-read processing and genome assembly were performed as previously described [11], [12]. Additionally, 23 previously published S. suis genomes (Ss_02, Ss_08, Ss_20, Ss_45, Ss_46, Ss_52, Ss_53, Ss_69, Ss_70, Ss_72, Ss_84, Ss_92, Ss_93, Ss_100, Ss_106, Ss_109, Ss_115, Ss_121, Ss_124, Ss_134, Ss_156, Ss_166, and Ss_167) were included [11], [12]. To check genome quality dRep bioinformatic tool (3.5.0 version) was used [16], applying the check-M step to evaluate the contamination and completeness of the genomes [17]. The threshold for contamination was 0–5% and for completeness 99–100%. An additional quality criterium proposed by [18] was applied (N50 > 30 kb). STs were determined using MLST 2.0 software [19]. AMR genes were identified with ABRicate using ResFinder and NCBI AMR gene database with a minimum of 80% of DNA identity and DNA coverage. ICEs and IMEs were identified with ICEScreen v1.3.1 [20] using pseudochromosomes as input [9] and were manually delimited in Geneious Prime version 2024.0.7 (Dotmatics). Comparative analyses of MGEs were performed with MAUVE [21]. MGEs were illustrated using EasyFig [22]. Prophages were detected with Phold v0.2.0. Recombination breakpoints were analyzed using RDP4, only recombination events positive to three of the seven detection methods applied (RDP, GENECONV, BootScan, MaxChi, Chimaera, 3Seq, and SiScan) and with a p-value <0.05 were considered significant [23].

2.4. Genetic constructions and preparation of mutants

Spontaneous rifampicin-resistant mutants, generated in S. suis strains Ss_45 and Ss_124, S. agalactiae strain SagS1, and S. pyogenes strain SpyS1 were performed as described [24] (extended in Text S1). For directed mutagenesis, overlapping PCR was used [25], using the PCR reactions described below. The three amplicons were purified with the FavorPrep™ GEL/PCR Purification Kit (Favorgen, Taiwan) and fused using the In-Fusion® HD Cloning Kit (Takara, Korea). The resulting hybridised fragment was used to transform S. suis with the ComS peptide [26], [27]. Transformants were selected on THA plates supplemented with the appropriate antibiotics and incubated at 37 °C under 5% CO₂ for 24–48 h.

2.5. Co-incubation experiments

Mating experiments were performed as reported [28] (detailed in Text S1). The S. suis strains Ss_20 and Ss_115 were used as donors; these strains were sensitive to spectinomycin, penicillin, and rifampicin, but resistant to erythromycin and tetracycline. The recipient strains were the spectinomycin-resistant S. suis P1/7∆gfp + (Saralegui et al., under revision), which is sensitive to erythromycin and tetracycline, the penicillin-resistant S. pneumoniae SpS1, and the spontaneous rifampicin-resistant S. agalactiae SaS1R1 and S. pyogenes SpyS1R2. To assess recombination between ICEs, the resulting transformants were tested by PCR as described below. The conjugation rate was estimated by dividing the number of colony-forming units (CFU) of the transconjugants by the CFU of the donor bacteria at the end of the conjugation experiments. For growth competition assays, the same mating method was used (further described in Text S1). Bacteriocin production was tested as described [28].

2.6. PCR amplification

PCR was used for four purposes in this study. First, to detect and co-localize the erm(B) and tet(O) genes using the Supreme NZYLong DNA polymerase kit (Nzytech, Portugal). Second, to discriminate between Streptococcus species during mating experiments by targeting species-specific genes, including gdh for S. suis [29], mecA for S. agalactiae, lytA for S. pneumoniae [30], and spy for S. pyogenes [31]. In S. suis-S. suis matings, the spectinomycin-resistance cassette was amplified to identify P1/7Δgfp+ [32]. Third, primers targeting ICE-specific regions (ICE45_rec_D, ICE124_rec_D, and ICE_rec_U) were used to analyse transformants. For the second and third purposes, a standard DNA Taq polymerase kit was used (Biotools, Spain). Finally, PCR was employed to generate directed mutants by replacing tet(O) with the cat gene using the High-Fidelity Phusion DNA polymerase (Thermo Fisher Scientific, USA). The primers are listed in Table S2. Each PCR reaction contained 0.4–0.5 μM of each primer, 200–500 μM dNTPs, 0.4–1 U of DNA polymerase, and the corresponding buffer. Amplification consisted of initial denaturation at 94 °C for 2–5 min, 35 cycles of 94 °C for 45 s, annealing the primer-specific temperature for 45 s (Table S2), and extension at 68–72 °C for 1 min/kilobase, followed by a final extension at 68–72 °C for 7–30 min. PCR products were separated on 0.7%–1% agarose gels stained with Green®Nucleic Acid Stain (Sigma-Aldrich, Germany), and sequenced at STABVida when required.

3. Results

3.1. Clinical S. suis isolates carry a diversity of MGEs

Our previous analysis of invasive Spanish swine S. suis isolates collected across Spain revealed high resistance rates (>90%) to tetracyclines, macrolides, and lincosamides, which were statistically associated with the tet(O) and erm(B) genes [12]. Both genes are often co-located on MGEs, mostly ICEs or IMEs, transferable by conjugation [10], [33]. Because our study aimed to demonstrate the transfer of AMR genes to other human-pathogenic species in natural environments, we focused on tet(O) and erm(B), given their high prevalence and potential mobility. PCR screening of 91 S. suis isolates positive for tet(O) and erm(B) confirmed their co-localization in 79 isolates, with amplicon sizes ranging from 3 kb to 20 kb (Table S3), suggesting that these genes are often carried on the same MGE. Then, genome sequencing of 34 positive isolates from different geographic origins and STs identified a total of 134 MGEs (Table S4 and expanded in Text S1). These included 62 ICEs from four families: Tn5252 (75.8%), Tn1549 (14.5%), TnGBS2 (8.1%), and ICESt3 (1.6%), and 87 IMEs from six families: PF01076 (35.6%), PF02486 (32.2%), PF01719 (12.6%), PF01719-PF00910 (8%), PHA00330 (6.9%), and PF13814 (4.6%). AMR genes were detected in 28 ICEs, 5 defective ICEs (dICEs), one partial ICE, 24 IMEs, and 6 defective IMEs (dIMEs) located within ICEs (Table 1). The ICEs/dICEs harbored up to six distinct AMR gene patterns, differing in order, orientation and content (Fig. 1B) while IMEs showed a similar gene composition. All AMR-carrying ICEs belonged to the Tn5252-family, and were inserted into rplL (29.4%), rumA (29.4%), mutT (23.5%), ADP ribose pyrophophatase (8.8%) or NTP pyrophosphohydrolase (8.8%) genes (Fig. 1A). AMR-carrying IMEs belonged to the PF01076 family, inserted into SNF2 (22/30) or peptidylprolyl isomerase (PPI) (7/30) genes. Comparative analysis of the Tn5252-ICEs harbouring tet(O) and/or erm(B) revealed 95.4–99.9% nucleotide identity over 32–100% of their sequence length, with genetic distance ranging from 0.33 to 0.99. 22 ICEs showed a genetic distance >0.85, while 12 ICEs were more divergent (<0.85). These findings highlight that S. suis harbors diverse MGEs, but those carrying tet(O) and erm(B) are exclusively Tn5252-family ICEs. Although these ICEs share extensive conserved regions also exhibit substantial sequence and gene cargo variability.

Table 1.

Characteristics of Mobile Genetic Elements (MGEs) carrying Antimicrobial Resistance (AMR) genes in the genomes of 34 S. suis and 11 S. agalactiae strains. AMR genes indicated in the [3′-5′] direction is marked with an asterisk, genes that are partial are marked with two asterisks. Integration site of MGEs is indicated at the end of the given name and unidentified integration sites in the genome sequence are denoted with a ^# symbol.

Isolate	Name of ICE	VirB4 hit	Integrase(s)	Relaxase family	Size of ICE (bp)	AMR genes in ICE	Name of nested IME	Integrase of IME	Relaxase family of IME	AMR genes in IME
S. suis
Ss_02	ICESsu_02_ Tn5252_rumA	AER19616	Serine recombinase (AGG64091)	MOBP (PF03432)	62,887	tet(O), erm(B)	dIMESsu_02_ PF01076_SNF2	Serine recombinase shorter	MOBV (PF01076)	tet(O)
Ss_08	ICESsu_08_ Tn5252_rplL	ADV69676	Tyrosine integrase (ABP92066)	MOBP (PF03432)	75,532	tet(O), erm(B), aadE, sat4, aph(3′)-III	IMESsu_08_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B), aadE, sat4, aph(3′)-III
Ss_20	ICESsu_20_ Tn5252_ SSU1797	ADV69676	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	71,711	tet(O), erm(B)	IMESsu_20_ PF01076_PPI	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_27	ICESsu_27_ Tn5252_ SSU1262	ABP89935	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	66,279	tet(O), aadE, erm(B)	dIMESsu_27_ PF01076_SNF2	–	–	tet(O), aadE, erm(B)
Ss_31	ICESsu_31_ Tn5252_rumA	AER19616	Serine recombinase (AGG64091)	MOBP (PF03432)	55,690	tet(O), erm(B)	–	–	–	–
Ss_45	ICESsu_45_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	6881	erm(B)*, tet(O), erm(B)	IMESsu_45_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_46	ICESsu_46_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	11,701	tet(O), erm(B)	IMESsu_46_ PF01076_PPI	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_50	dICESsu_50_ Tn5252_mutT	Partial	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	64,633	tet(O), erm(B)	IMESsu_50_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_52	ICESsu_52_ Tn5252_rplL	ABP89935	Tyrosine integrase (CAZ51585)	MOBP (PF03432)	66,330	tet(O), erm(B)	IMESsu_52_ PF01076_PPI	Serine recombinase (AER15058)	MOBV (PF01076)	tet(O)
Ss_53	dICESsu_53_ Tn5252_rplL	ABP89935	Tyrosine integrase (ABP92066)	MOBP (PF03432)	65,433	tet(O), erm(B)	IMESsu_53_ PF01076_PPI	Serine recombinase (AER15058)	MOBV (PF01076)	tet(O)
Ss_61	ICESsu_61_ Tn5252_rplL	ABP89935	Tyrosine integrase (CAZ51585)	MOBP (PF03432)	71,742	tet(O), erm(B)	IMESsu_61_ PF01076_PPI	Serine recombinase (AER15058)	MOBV (PF01076)	tet(O)
Ss_64	ICESsu_64_ Tn5252_rumA	AER19616	Serine recombinase (AGG64091)	MOBP (PF03432)	62,886	tet(O), erm(B)	dIMESsu_64_ PF01076_SNF2	Serine recombinase shorter	MOBV (PF01076)	tet(O)
Ss_69	Partial ICESsu_69_ Tn5252_rplL	ABP89935	–	–	48,080	erm(B), tet(O)	IMESsu_69_ PF01076_SNF2	Serine recombinase (AER15058)	MOBV (PF01076)	tet(O)
Ss_70	dICESsu_70_ Tn5252_rplL	Partial	Tyrosine integrase (AER17235)	MOBP (PF03432)	82,849	tet(O), aadE*, erm(B)	dIMESsu_70_ PF01076_SNF2	Serine recombinase shorter	MOBV (PF01076)	tet(O), aadE*, erm(B)
Ss_72	ICESsu_72_ Tn5252_rplL	ADV69676	Tyrosine integrase (CCW38101)	MOBP (PF03432)	68,265	tet(O), erm(B)	IMESsu_72_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_81	ICESsu_81_ Tn5252_rumA	AER19616	Serine recombinase (AGG64091)	MOBP (PF03432)	62,887	tet(O), erm(B)	dIMESsu_81_ PF01076_SNF2	Serine recombinase shorter	MOBV (PF01076)	tet(O)
Ss_84	ICESsu_84_ Tn5252_mutT	AER17274	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	68,902	tet(O), erm(B)	IMESsu_84_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_92	ICESsu_92_ Tn5252_rplL	ADV69676	Tyrosine integrase (ABP92066)	MOBP (PF03432)	65,088	tet(O), erm(B)	dIMESsu_92_ PF01076_SNF2	Serine recombinase shorter	MOBV (PF01076)	tet(O), erm(B)
Ss_93	ICESsu_93_ Tn5252_ SSU1797	ADV69676	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	66,630	tet(O), erm(B)	IMESsu_93_ PF01076#	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_100	ICESsu_100_ Tn5252_mutT	AER17274	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	66,159	tet(O), erm(B)	IMESsu_100_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_105	ICESsu_105_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	67,845	tet(O), erm(B)*	IMESsu_105_ PF01076_PPI	Serine recombinase (AER17248)	MOBV (PF01076)	tet(O), erm(B)*
Ss_106	ICESsu_106_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	73,848	tet(O), erm(B)	IMESsu_106_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_109	ICESsu_109_ Tn5252_ SSU1797	AER15081	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	70,309	tet(O), erm(B)	IMESsu_109_ PF01076_PPI	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_110	ICESsu_110_ Tn5252_mutT	ADV69676	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	66,112	tet(O), erm(B)	IMESsu_110_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_115	ICESsu_115_ Tn5252_ SSU1262	ADV69676	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	65,505	tet(O), erm(B)	–	–	–	–
Ss_121	ICESsu_121_ Tn5252_mutT	ADV69676	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	70,401	erm(B), tet(O)	IMESsu_121_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O)
Ss_124	ICESsu_124_ Tn5252_rplL	AER15081	Tyrosine integrase (ABP92066)	MOBP (PF03432)	75,451	tet(O), erm(B), aadE, sat4, aph(3′)-III	IMESsu_124_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B), aadE, sat4, aph(3′)-III
Ss_134	dICESsu_134_ Tn5252_ SSU1262	ABP89935	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	66,143	tet(O), aadE, erm(B)	–	–	–	–
Ss_146	ICESsu_146_ Tn5252_mutT	AER17274	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	66,159	tet(O), erm(B)	IMESsu_146_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_156	ICESsu_156_ Tn5252_mutT	AER17274	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	66,163	tet(O), erm(B)	IMESsu_156_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_160	ICESsu_160_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	72,867	tet(O), erm(B)	IMESsu_160_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Ss_165	ICESsu_165_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	68,486	erm(B), tet(O)	IMESsu_165_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O)
Ss_166	dICESsu_166_ Tn5252_mutT	Partial	Triplet of serine recombinases (AGZ23088, AGZ23089, AGZ23090)	MOBP (PF03432)	92,082	tet(O), erm(B)	–		–	–
Ss_167	ICESsu_167_ Tn5252_rplL	ADV69676	Tyrosine integrase (ABP92066)	MOBP (PF03432)	64,316	tet(O)	IMESsu_167_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O)
S. agalactiae
Sa_26	ICESag_26_ Tn916_guaA	CBJ22573	Tyrosine integrase (CBJ22584)	MOBT (PF02486)	21,853	tet(M)	–	–	–	–
	ICESag_26_ Tn5252_rplL	ABP89935	Tyrosine integrase (ADX24462)	MOBP (PF03432)	71,022	tet(O), erm(B)	IMESag_26_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Sa_37	ICESag_37_ Tn916_guaA	CBJ22573	Tyrosine integrase (CBJ22584)	MOBT (PF02486)	21,864	tet(M)	–	–	–	–
	ICESag_37_ Tn5252_rplL	ABP89935	Tyrosine integrase (ADX24462)	MOBP (PF03432)	71,022	tet(O), erm(B)	IMESag_37_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Sa_44	ICESag_44_ Tn916_guaA	CBJ22573	Tyrosine integrase (CBJ22584)	MOBT (PF02486)	21,853	tet(M)	–	–	–	–
	ICESag_44_ Tn5252_rplL	ABP89935	Tyrosine integrase (ADX24462)	MOBP (PF03432)	71,022	tet(O), erm(B)	IMESag_44_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Sa_48	ICESag_48_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	70,880	tet(O), erm(B)	IMESag_48_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Sa_56	ICESag_56_ Tn5252_rplL	CCF02711	Tyrosine integrase (CCW38101)	MOBP (PF03432)	56,990	mef(A), mrs(D)	–	–	–	–
	ICESag_56_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	71,171	tet(O), aadE, sat4**, aph(3′)-III, erm(B), aadE	dIMESag_56_ PF01076_PPI	Serine recombinase (AER15058)	–	tet(O), aadE, sat4**, aph(3′)-III, erm(B), aadE
Sa_75	ICESag_75_ Tn5252_rplL	ADV69676	Tyrosine integrase (ADX24462)	MOBP (PF03432)	69,483	tet(O), erm(B)	IMESag_75_ PF01076_SNF2	Serine recombinase (ABF36192)	MOBV (PF01076)	tet(O), erm(B)
Sa_79	ICESag_79_ Tn5252_rplL	ADV69676	Tyrosine integrase (ADX24462)	MOBP (PF03432)	68,772	tet(O)	dIMESag_79_ PF01076_SNF2	Serine recombinase shorter	MOBV (PF01076)	tet(O)
	ICESag_79_ ICESt3_tRNAlys	EAO72173	Tyrosine integrase xerC	MOBT (PF02486)	38,752	lsa(C)	IMESag_79_ PF02486_oriT	Tyrosine integrase	MOBT (PF02486)	lsa(C)
Sa_82	ICESag_82_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	73,408	tet(O), aadE, sat4**, aph(3′)-III, erm(B), aadE	dIMESag_82_ PF01076_PPI	Serine recombinase (AER15058)	–	tet(O), aadE, sat4**, aph(3′)-III, erm(B), aadE
Sa_83	ICESag_83_ Tn5252_rplL	ADV69676	Tyrosine integrase (ADX24462)	MOBP (PF03432)	68,772	tet(O)	dIMESag_83_ PF01076_SNF2	Serine recombinase shorter	MOBV (PF01076)	tet(O)
Sa_85	ICESag_85_ Tn5252_rplL	CCF02711	Tyrosine integrase (CCW38101)	MOBP (PF03432)	56,909	mef(A), mrs(D)	–		–	–
	ICESag_85_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	73,669	tet(O), aadE, sat4**, aph(3′)-III, erm(B), aadE	dIMESag_85_ PF01076_PPI	Serine recombinase (AER15058)	–	tet(O), aadE, sat4**, aph(3′)-III, erm(B), aadE
Sa_86	ICESag_86_ Tn916	BAK30694	Tyrosine integrase (EIK41785)	MOBT (PF02486)	18,038	tet(M)	–		–	–
	ICESag_86_ Tn5252_rumA	ADV69676	Serine recombinase (AGG64091)	MOBP (PF03432)	69,637	tet(O), erm(B)	dIMESag_86_ PF01076_SNF2	Serine recombinase shorter	MOBV (PF01076)	tet(O), erm(B)

Abbreviations: SNF2; encoding a putative helicase protein, PPI: peptidylprolyl isomerase, “-“: no IME inserted in ICE.

Fig. 1.

Comparison of the Integrative and Conjugative Elements (ICEs) in S. suis isolates belonging to the Tn5252-family. (A) Nucleotide comparative in 8 representative ICEs. Arrows represent genes and their orientation within the ICE sequence. Relevant genes involved in the conjugation and integration are shown in different colours, and AMR genes are indicated in red, lantibiotic immunity proteins in violet, bacteriocin production genes in light blue, IS transposases in dark green, and transposons in orange, as declared in the inset. Grey arrows represent the integration genes of Integrative and Mobilizable Elements (IMEs). Similar regions and percentages of identity between ICEs are indicated below in grayscale. Integration sites are specified in the names of the ICEs. (B) AMR genes (grey) carried by the different types of mobile genetic elements (coloured) detected in S. suis isolates. ICEs (left panel) and IMEs (right panel) are shown in different graphs. Ribbons are wider when more elements contain the AMR gene. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.2. Tn5252-family ICEs are present in other streptococci isolated in Spain

Aragón is a leading region in Spanish pig production producing around 40 million pigs/year between 2016 and 2021, while hosting 1.3 million people. Thus, this region is a suitable environment to detect whether AMR gene transfer occurred between S. suis and other human pathogens. To investigate this, we analyzed tetracycline and erythromycin resistance in clinical streptococci recovered at the region's major Hospital (Hospital Universitario Miguel Servet, Zaragoza) between 2019 and 2021 (matching the S. suis collection period), including 539 S. pneumoniae, 1519 S. agalactiae, and 365 S. pyogenes isolates. S. pneumoniae exhibited moderate AMR rates to both antibiotics (15–30%), while S. agalactiae revealed higher AMR rates to tetracycline (>30%), and S. pyogenes exhibited low AMR rates to all antibiotics tested (Table S5).

PCR screening of representative resistant S. agalactiae and S. pneumoniae isolates for tet(O) and erm(B) evidenced 25% of S. pneumoniae isolates carried erm(B), and 48% and 52% of S. agalactiae isolates carried both genes, respectively (Table S1). Eleven S. agalactiae carried both genes co-located, presumably within the same MGE (Table S3, Fig. 2A, and extended in Text S1). These isolates belonged to five unrelated STs: ST498 (Sa_26, Sa_37, Sa_44), ST17 (Sa_56, Sa_82, Sa_85), ST28 (Sa_48 and Sa_86), ST529 (Sa_79, and Sa_83), and ST196 (Sa_75). Genome sequencing and MGEs analysis identified 36 ICEs from 6 families: Tn5252 (36.1%), ICESt3 (33.3%), TnGBS2 (13.9%), Tn916 (11.1%), Tn1549 (2.8%), and TnGBS1 (2.8%), of which Tn5252, Tn916 and ICESt3 carried AMR genes. Thirty-nine IMEs from six families were identified at different frequencies: PF01719 (28.9%), PF01076 (26.3%), PF02486 (23.7%), PF01076_PF02486 (7.9%), PHA00330 (7.9%), and PF02407 (5.3%). Twelve IMEs contained AMR genes, corresponding to PF01076 and PF02486 families(Fig. 2B). The tet(O) and erm(B) genes were identified in 11 Tn5252-family ICEs inserted into rplL or rumA genes. Comparative analysis of these Tn5252-ICEs showed a 96.5–99% nucleotide identity over 67–100% of their length (Fig. 2A) and genetic distance ranging from 0.61 to 0.99. Isolates Sa_26, Sa_37 and Sa_44 (ST498) carried identical ICEs suggesting vertical transmission. These findings show that S. agalactiae harbors Tn5252-family ICEs with tet(O)-erm(B), as the S. suis collection. Comparative analysis of Tn5252-family ICEs found in both species revealed different levels of genetic proximity and sequence identify. Interestingly, ICESsu_92_Tn5252_rplL of S. suis Ss_92 showed 98.6% of sequence identity over 64,984 bp (99.8% of its length) with ICE_Tn5252_rplL in S. agalactiae Sa_26, Sa_37, and Sa_44 (Fig. 3A), and 98% identity over 61,590 bp (94.6% of its length) with ICE_Tn5252_rplL in Sa_75 (Fig. 3A). Other Tn5252-ICEs of S. suis isolates such as Ss_31, Ss_165 and Ss_124 showed slightly lower identity (97.5–97.9%) with Tn5252-ICEs of S. agalactiae isolates Sa_86, Sa_48, and Sa_26 compared with the previous ones (Fig. 3B). Together, at least six of eleven S. agalactiae isolates carried Tn5252-family ICEs with substantial sequence identity (96.8–98.6%) over >91% of the element compared with ICEs found in S. suis. Notably, the most similar ICEs were identified in S. suis and S. agalactiae isolates recovered in Aragón, suggesting interspecies horizontal transfer of these elements and subsequent diversification.

Fig. 2.

(A) Comparison of the genetic organization of six representative Integrative and Conjugative Elements (ICEs) of the Tn5252-family identified in S. agalactiae isolates carrying antibiotic resistance (AMR) genes. Colour coding for genes is the same used in Fig. 1. Similar regions and percentages of identity between ICEs are indicated below in grayscale. Integration sites are specified in the names of the ICEs. (B) AMR genes (grey) carried by the different types of mobile genetic elements (coloured) detected in S. agalactiae isolates. ICEs (left panel) and IMEs (right panel) are shown in different graphs. Ribbons are wider when more elements contain the AMR gene.

Fig. 3.

Comparison of most alike Integrative and Conjugative Elements (ICEs) carrying tet(O) and erm(B) found between S. suis and S. agalactiae isolates from Aragón. The comparison includes (A) the S. suis ICEs with the highest percentage of identity to S. agalactiae ICEs, including ICESag_37_Tn5252_rplL, ICESsu_92_Tn5252_rplL, and ICESag_75_Tn5252_rplL, and (B) ICEs with an intermediate percentage of identity, such as ICESag_86_Tn5252_rumA, ICESsu_165_Tn5252_rumA, and ICESag_48_Tn5252_rumA. Colour coding for genes is the same used in Fig. 1. Similar regions and percentages of identity between ICEs are indicated below in grayscale.

3.3. S. suis can transfer AMR genes via MGEs to other Streptococcus species in vitro

S. pneumoniae and S. pyogenes lacked the erm(B) and tet(O) commonly found Tn5252-family ICE in S. suis, suggesting less frequent AMR gene exchange between these streptococci than with S. agalactiae. To investigate the potential of S. suis to transfer MGEs, we conducted mating experiments using various donor/recipient ratios. Donor strains were S. suis isolates Ss_20 and Ss_115, which harbour ICE_Tn5252 inserted into the SSU1797 and SSU1262 locus, respectively. Recipient strains included isolates from S. suis P1/7Δgfp+, S. agalactiae SaS1R1, S. pneumoniae SpS1, and S. pyogenes SpyS1R2. Conjugation rates varied significantly depending on the recipient species and the donor/recipient ratio (Fig. 4A), obtaining higher conjugation rates when P1/7Δgfp + was recipient. To explore whether interspecies-growth competition caused these differences, CFUs of all strains were determined after individual and co-incubated cultures. CFUs of Ss_20 and Ss_115 were significantly (p < 0.05) reduced when co-incubated with other streptococcal species, except for Ss_115 with SpS1 (Fig. 4B). Conversely, CFUs of recipients were unaffected, except for P1/7Δgfp + co-incubated with Ss_115 (Fig. 4B). No bacteriocin-mediated growth inhibition was observed in any case. The prophage of Ss_20 and three prophages of Ss_115 encode holin products with lytic function. These prophages might be activated during co-incubation, causing donor cell death. Our results demonstrate that S. suis can transfer MGEs to other streptococci in vitro, although conjugation efficiency is recipient-dependent.

Fig. 4.

Mating experiments with different Streptococcus species. (A) Conjugation rates obtained from mixtures with donor strains Ss_20 and Ss_115 of S. suis with recipient strains P1/7Δgfp + of S. suis, SaS1R1 of S. agalactiae, SpS1 of S. pneumoniae, and SpyS1R2 of S. pyogenes in 4 different donor/recipient (D/R) ratios. (B) Post-incubation strains quantification. Left panel shows bacterial counts after incubation alone or with donor and recipient strains. Right panel shows the recipient bacterial counts. Data are the median and range of three independent assays. Significant differences (p < 0.05, unpaired t-test) as compared to the control group (alone) are indicated with one asterisk.

3.4. ICE diversity can be generated by recombination events

Tn5252-family ICEs from S. suis contain shared regions but display high variability in gene cargo content. We hypothesized this results from recombination events between ICEs. To investigate this, we engineered a mutant in strain P1/7Δgfp+, which harbors ICESsu_115_Tn5252_SSU1262 (P1/7ICE115), by substituting tet(O) with a chloramphenicol-resistance cassette (cat) (P1/7ICE115ΔtetO). Genomic DNA of this mutant was used to transform rifampicin-resistant isolates Ss_45r and Ss_124r, which harbour ICESsu_45_Tn5252_rumA and ICESsu_124_Tn5252_rplL, respectively. Three transformants of Ss_45r (Ss_45r_Tf25, Ss_45r_Tf29, and Ss_45r_Tf33) and three from Ss_124r (Ss_124r_Tf5, Ss_124r_Tf8, and Ss_124r_Tf10) were tested by PCR and detailed results are provided in Figs. S1B-C and Text S1. Briefly, PCR screening suggested that the ICE of Ss_45r_Tf25, Ss_45r_Tf33, Ss_124r_Tf5, and Ss_124r_Tf10 had ICEs with upstream- and downstream-tet(O) regions identical to the recipient ICE, whereas ICEs of clones Ss_45r_Tf29 and Ss_124r_Tf8 had downstream-tet(O) region matching the recipient, but the upstream-tet(O) region of donor ICE. Whole-genome sequencing of Ss_45r_Tf29, Ss_45r_Tf33, and Ss_124r_Tf10 confirmed structural differences in the ICEs of the three transformants (Fig. 5A-C detailed in Text S1). Analysis with RDP4 detected significant recombination points (p-value of 1 × 10⁻³⁰) in the three ICEs (orange coloured in Fig. 5A-C). Notably, each transformant carried a uniquely composed Tn5252-family ICE, demonstrating that distinct recombination events can generate ICE diversity.

Supplementary Fig. S1.

(A) Schematic representation of DNA fragments a, b, and c for screening recombinant clones. Portions of the Integrative and Conjugative Element (ICE) from the recipient strains Ss_45r and Ss_124r, and the donor strain P1/7ICE115ΔtetO, are shown. The expected length of the fragment a1 is 9338 base pairs (bp) and of the fragment a2 is 5209 bp. The expected length of the fragment b1 is 7654 bp and of the fragment b2 is 5259 bp. The expected length of the fragment c in Ss_45r and Ss_124r is of 3707 bp and 3654 bp, respectively. No amplification is expected for this fragment in P1/7ICE115ΔtetO. Genes are shown as grey arrows, antimicrobial resistance genes (AMR) are indicated with different colours (see legend). (B—C) Agarose gels showing PCR products obtained in different strains using primers for (B) fragment a, (C) fragment b, and (D) fragment c. Templates are labelled as follows: line 1) Ss_45r_Tf25, line 2) Ss_45r_Tf29, line 3) Ss_45r_Tf33, line 4) Ss_45r, line 5) P1/7ICE115ΔtetO, line 6) Ss_124r_5, line 7) Ss_124r_8, line 8) Ss_124r_10, and line 9) Ss_124r. Abbreviations: ICE, Integrative and Conjugative Elements. Neg, ultra-pure water. Interpretation: To analyse the downstream-tet(O) region, different primers sets were designed for the transformants of Ss_45r and Ss_124r, yielding fragments a and b. If the fragment length matched that of the recipient strain, the fragments were denominated as a1 and b1. Conversely, if the fragment length matched that of the donor, they were referred as a2 and b2 (Fig. S1A). All Ss_45r transformants yielded fragment a2 (Fig. S1B). Similarly, all Ss124r transformants yielded fragment b2 (Fig. S1C). These results suggest that the downstream region in all transformants shared the same organization as the donor strain. In contrast, analysis of the upstream tet(O) region (fragment c) revealed PCR amplification only in recipient-like sequences (Fig. S1A). All 6 transformants were tested for the presence of fragment c (Fig. S1D). Clones Ss_45r_Tf25, Ss_45r_Tf33, Ss_124r_Tf5, and Ss_124r_Tf10 yielded fragment c, consistent with the recipient strains, but clones Ss_45r_Tf29 and Ss_124r_Tf8 were negative (Fig. S1D).

Fig. 5.

Mosaic organization of Integrative and Conjugative elements (ICEs) generated by recombination within ICEs. Genetic rearrangements (A, B) in the ICE of the transformants Ss_45r (C) and Ss_124r after incubation with P1/7ICE115ΔtetO. For all panels, the recipient and donor strain are located above and below the recombinant strain, respectively. Gene colour coding follows the scheme used in Fig. 1, with additional arrows in pink indicating the erm(B) gene, maroon arrows indicating tet(O), and green arrows indicating the cat gene. The most probable recombination area is highlighted in orange. Similar regions and percentages of identity between ICEs are indicated below in grayscale. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

While plasmid-mediated spread is a well-established mechanism for AMR dissemination from farms to humans, the contribution of conjugative chromosomal elements is less well understood. S. suis, a porcine pathogen highly prevalent and a multidrug-resistant bacterium, is considered a reservoir of AMR genes. This study provides, for the first time, substantial evidence that combines comparative genomics of clinical strains from the same geographic region with in vitro conjugation assays, supporting the conclusion that this bacterium transfers AMR genes directly to human-pathogenic streptococci.

Previous studies have proved in vitro that S. suis can transfer AMR genes to other species, including S. pyogenes [34], [35], [36], [37], S. agalactiae [37], [38], S. pneumoniae [35], [36], S. oralis [36], and S. thermophilus [39]. These results were confirmed in our work using clinical isolates. We observed higher transfer rates between S. suis strains than with other streptococci, consistent with a prior report [35]. However, the efficiency of the transfer was diverse depending on the recipient species and occurred under specific D/R ratios. This indicates that horizontal gene transfer is highly dependent of the context, including the bacterial density and interspecies competition. Furthermore, our results showed reduced viability of donor strains during co-incubation with the recipients, this phenomenon could be caused by inter-strain growth inhibition systems, including bacteriocin production, previously reported in several streptococci species [40], [41], [42], or by the activation of lytic prophages [43]. Additionally, the activity of restriction-modification systems and clustered regularly interspaced short palindromic repeats defences may prevent ICE insertion and reduce transfer efficiency as demonstrated in S. agalactiae [44]. Thus, AMR transfer from S. suis to other streptococci via conjugation may be limited by different factors. This can explain the fact that most previous evidence of interspecies AMR gene transfer involving S. suis comes from comparative genomic analyses of clinical isolates rather than direct experimental demonstration in natural environments. As conjugation in vitro is hard to reproduce, its frequency in vivo it is assumed to be low. For instance, Martel, et al. (2005) [36] detected erm(B) and tet(O) in S. suis isolates and other streptococcal species of both human and animal origin in Belgium, while the tet(O/W/32/O) gene was identified in pig isolates of S. suis in Italy [45] and in a S. gallolyticus isolate from a human patient in Germany [46]. Also, Huang et al. (2016) [10] identified ICEs of the family Tn5252 carrying tet and erm gene variants in seven Chinese S. suis isolates that were present in streptococcal species deposited in public databases [10]. However, reconciling these comparative analyses with interbacterial interaction required for conjugation is challenging, as these elements show limited sequence homology and ICEs of the same family and AMR genes are widely distributed across streptococci species. Furthermore, clinical isolates and public genomes sharing these elements are geographically very distant. Here, we detected a large diversity of conjugative elements, including Tn5252-family ICEs, in our S. suis collection. These elements carry tet(O) and erm(B) but show highly variable lengths and a mosaic genetic pattern. Remarkably, we detected S. suis isolates harbouring Tn5252-family ICEs almost identical to those found in S. agalactiae isolates, and both species were recovered from the same region and time period. To the best of our knowledge, this study shows, for the first time, substantial evidence of transfer between S. suis and other species in a within a shared epidemiological context involving both animal and human populations.

S. suis can colonize the mucosa of several animal species and behave as a commensal. Previous reports have detected this bacterium in high-risk populations, including pig workers, meat inspectors, veterinarians, slaughterhouse workers, or butchers [47], [48], [49], [50]. While the duration of colonization is unknown, it can persist for at least three weeks [51]. In contrast, S. agalactiae is a natural component of the human microbiota. We hypothesize that in Aragón, S. suis colonized humans and, by sharing an ecological niche with S. agalactiae, transferred AMR genes via Tn5252-family ICEs. However, we cannot determine whether the exchange occurred directly on these strains or through an intermediate host. A interestingly observation is that three out of the six S. agalactiae isolates sharing an ICE with S. suis isolates belonged to the same ST but were from different patients. This suggests that a common ancestor acquired the ICE from S. suis, with subsequent vertical dissemination. Slight sequence differences indicate that these ICEs evolved independently after transfer. The remaining isolates belonged to different STs and carry ICEs with slightly lower identity, suggesting independent transfer events either from S. suis or S. agalactiae strains. Additionally, some isolates show a lower identity with ICEs of our S. suis collection, probably reflecting older transfer events followed by diversification.

Tn5252-family ICEs in S. suis revealed a mosaic gene organization with variable AMR genes composition. The origin of the variability in the adaptive module of ICEs was attributed to different mechanisms [52], including recombination, transposon activity, or acquisition of new ICEs. Marini et al. (2015) reported a hybrid ICE generated by recombination between S. suis ICESsu32457 and S. agalactiae ICESa2603 probably transferred by conjugation [38]. However, conjugation leads, generally, to the acquisition of a complete ICE or duplication. Despite sequence similarity with ICEs present in the recipient strain, site specific recombination is more efficient than homologous recombination, and intracellular intermediates (circled double stranded DNA) are not substrates for RecA. Thus, the acquisition of the ICE rather than recombination will be favored during conjugation. This does not exclude that ICEs can be recognized by self-protecting mechanisms, and the subsequent products can then be recognized by the homologous recombination system. We proposed that transformation rather than conjugation can be a key driver of ICE diversification, likely promoting the diversity found in Tn5252-family ICEs of S. suis (Fig. 1A). This was proved here by experiments using DNA from strains carrying reporter genes within Tn5252-family ICEs and further genome sequencing of recombinants. This phenomenon was also reported in other streptococcal species, including in Tn916-related ICEs of S. pneumoniae [53], ICE_Tn5252 of S. agalactiae [38], as well as within ICEs of Enterococcus faecalis [54]. The resulting hybrids with new gene content were transferred by conjugation to other streptococci, demonstrating that gene exchange did not affect the transfer mechanism, generating thus a new transferable AMR gene combination. Comparative analysis of the frequency of these mechanisms, which was not addressed in this study, would help clarify their relative contribution to AMR diversification.

5. Conclusions

S. suis harbors a large repertoire of conjugative MGEs, which can carry multiple AMR genes and disseminate multi-drug resistance. The AMR genes patterns within a MGEs is variable and it can be generated through recombination events. These new combinations can eventually be transferred to other streptococcal species, generating multi-resistant strains in a single step. Our findings emphasize the need for integrated One Health approaches to reduce the spread of AMR.

Routine genomic monitoring of mobile genetic elements in both animal and human isolates could enable early detection of cross-species transmission events. In addition, targeted interventions such as antimicrobial stewardship programs in pig production, routine genomic surveillance of mobile genetic elements across veterinary and clinical sectors, or strict biosecurity measures to reduce animal to human transmission should be prioritized.

The following is the supplementary data related to this article.

Supplementary Text S1

Expanded description of material and methods, and results.

Supplementary Table S1

Strains and clinical isolates used in this study.

Abbreviations: ST: Sequence type, ERY: Erythromycin, CLIN: Clindamycin, TET: Tetracycline, NT: Non-typable, AMP: Ampicillin

Supplementary Table S2

Primers used in the study. The size of the expected PCR products and annealing temperature (T_A) for each PCR reaction is also indicated. Abbreviations: bp: base pairs, T_A: annealing temperature, AMR: Antimicrobial Resistance.

Supplementary Table S3

Molecular sizes (kb) obtained by PCR for the co-localization of tet(O) and erm(B) genes in 91 S. suis isolates and 11 S. agalactiae isolates.

Supplementary Table S4

Mobile Genetic Elements identified in the 34 genomes of S. suis and 11 genomes of S. agalactiae. The family of the Integrative and Conjugative Elements (ICEs) or Integrative and Mobilizable Elements (IMEs) is indicated for each MGE found in every isolate.

Supplementary Table S5

Prevalence of antimicrobial resistance to different antibiotics in isolates of S. pneumoniae, S. agalactiae, and S. pyogenes from human patients at Miguel Servet University (Zaragoza, Spain) between 2019 and 2021.

CRediT authorship contribution statement

Cristina Uruén: Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation. María José Lavilla: Writing – review & editing, Methodology, Investigation. Virginie Libante: Writing – review & editing, Validation, Supervision, Methodology, Investigation. Clara M. Marín: Writing – review & editing, Methodology, Investigation. Antonio Rezusta: Writing – review & editing, Methodology, Investigation. Sophie Payot: Writing – review & editing, Supervision, Software, Resources, Funding acquisition, Conceptualization. Jesús Arenas: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Data curation.

Ethical approval

No ethical approval is required.

Funding

This work received funding from Gobierno de Aragón (Department of I + D + I project in priority lines, Grant agreement LMP58_21), and Ministerio de Ciencia e Innovación/Agencia Española de Investigación MCIN/AEI/10.13039/501100011033, as appropriate, by ERDF A way of making Europe by the European Union or by the European Union NextGeneration EU/PRTR (Grant agreements PID2020-114617RB-100 and PID2023-146823OB-I00). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Glossary

AMR

Antimicrobial Resistance

MGE

Mobile Genetic Element

ICEs

Integrative and Conjugative Elements

IMEs

Integrative and Mobilizable Elements

ST

Sequence Type

MLST

Multi-Locus Sequence Type

THB

Todd-Hewitt Broth

THA

THB with 15% of Agar

OD₆₀₀

Optical Density at 600 nm

CFUs

Colony Forming Units

dICE

defective ICE

dIME

defective IME

PPI

Peptidylprolyl isomerase

Data availability

The datasets used in this study are available online in the database of NBCI under the bioprojects PRJNA1037519 and PRJNA1037513. Sequence of ICEs mutated during this study can be found below: ICESsu_115_Tn5252_SSU1262ΔtetO (PX363404), ICESsu_45_Tf29_Tn5252_rumA (PX363405), ICESsu_45_Tf33_Tn5252_rumA (PX363406), and ICESsu_124_Tf10_Tn5252_rplL (PX363407).