Exploration of DNA processing features unravels novel properties of ICE conjugation in Gram-positive bacteria.

Integrative and conjugative elements (ICEs) are important drivers of horizontal gene transfer in prokaryotes. They are responsible for antimicrobial resistance spread, a major current health concern. ICEs are initially processed by relaxases that recognize the binding site of oriT sequence and nick at a conserved nic site. The ICESt3/Tn916/ICEBs1 superfamily, which is widespread among Firmicutes, encodes uncanonical relaxases belonging to a recently identified family called MOBT. This family is related to the rolling circle replication initiators of the Rep_trans family. The nic site of these MOBT relaxases is conserved but their DNA binding site is still unknown. Here, we identified the bind site of RelSt3, the MOBT relaxase from ICESt3. Unexpectedly, we found this bind site distantly located from the nic site. We revealed that the binding of the RelSt3 N-terminal HTH domain is required for efficient nicking activity. We also deciphered the role of RelSt3 in the initial and final stages of DNA processing during conjugation. Especially, we demonstrated a strand transfer activity, and the formation of covalent DNA-relaxase intermediate for a MOBT relaxase.

Impact of Cell Surface Molecules on Conjugative Transfer of the Integrative and Conjugative Element ICESt3 of Streptococcus thermophilus.

Integrative conjugative elements (ICEs) are chromosomal elements that are widely distributed in bacterial genomes, hence contributing to genome plasticity, adaptation, and evolution of bacteria. Conjugation requires a contact between both the donor and the recipient cells and thus likely depends on the composition of the cell surface envelope. In this work, we investigated the impact of different cell surface molecules, including cell surface proteins, wall teichoic acids, lipoteichoic acids, and exopolysaccharides, on the transfer and acquisition of ICESt3 from Streptococcus thermophilus The transfer of ICESt3 from wild-type (WT) donor cells to mutated recipient cells increased 5- to 400-fold when recipient cells were affected in lipoproteins, teichoic acids, or exopolysaccharides compared to when the recipient cells were WT. These mutants displayed an increased biofilm-forming ability compared to the WT, suggesting better cell interactions that could contribute to the increase of ICESt3 acquisition. Microscopic observations of S. thermophilus cell surface mutants showed different phenotypes (aggregation in particular) that can also have an impact on conjugation. In contrast, the same mutations did not have the same impact when the donor cells, instead of recipient cells, were mutated. In that case, the transfer frequency of ICESt3 decreased compared to that with the WT. The same observation was made when both donor and recipient cells were mutated. The dominant effect of mutations in donor cells suggests that modifications of the cell envelope could impair the establishment or activity of the conjugation machinery required for DNA transport.IMPORTANCE ICEs contribute to horizontal gene transfer of adaptive traits (for example, virulence, antibiotic resistance, or biofilm formation) and play a considerable role in bacterial genome evolution, thus underlining the need of a better understanding of their conjugative mechanism of transfer. While most studies focus on the different functions encoded by ICEs, little is known about the effect of host factors on their conjugative transfer. Using ICESt3 of S. thermophilus as a model, we demonstrated the impact of lipoproteins, teichoic acids, and exopolysaccharides on ICE transfer and acquisition. This opens up new avenues to control gene transfer mediated by ICEs.

Surface proteins involved in the adhesion of Streptococcus salivarius to human intestinal epithelial cells.

The adhesion properties of 14 Streptococcus salivarius strains to mucus (HT29-MTX) and non-mucus secreting (Caco-2/TC7) human intestinal epithelial cells were investigated. Ability to adhere to these two eukaryotic cell lines greatly differs between strains. The presence of mucus played a major factor in adhesion, likely due to high adhesiveness to mucins present in the native human mucus layer covering the whole cell surface. Only one S. salivarius strain (F6-1), isolated from the feces of a healthy baby, was found to strongly adhere to HT-29 MTX cells at a level comparable to that of Lactobacillus rhamnosus GG, a probiotic strain considered to be highly adherent. By sequencing the genome of F6-1, we were able to identify 36 genes encoding putative surface proteins. Deletion mutants were constructed for six of them and their adhesion abilities on HT-29 MTX cells were checked. Our study confirmed that four of these genes encode adhesins involved in the adhesion of S. salivarius to host cells. Such adhesins were also identified in other S. salivarius strains.

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer.

Integrative and conjugative elements (ICEs) are widespread chromosomal mobile genetic elements which can transfer autonomously by conjugation in bacteria. Thirteen ICEs with a conjugation module closely related to that of ICESt3 of Streptococcus thermophilus were characterized in Streptococcus salivarius by whole-genome sequencing. Sequence comparison highlighted ICE evolution by shuffling of 3 different integration/excision modules (for integration in the 3' end of the fda, rpsI, or rpmG gene) with the conjugation module of the ICESt3 subfamily. Sequence analyses also pointed out a recombination occurring at oriT (likely mediated by the relaxase) as a mechanism of ICE evolution. Despite a similar organization in two operons including three conserved genes, the regulation modules show a high diversity (about 50% amino acid sequence divergence for the encoded regulators and presence of unrelated additional genes) with a probable impact on the regulation of ICE activity. Concerning the accessory genes, ICEs of the ICESt3 subfamily appear particularly rich in restriction-modification systems and orphan methyltransferase genes. Other cargo genes that could confer a selective advantage to the cell hosting the ICE were identified, in particular, genes for bacteriocin synthesis and cadmium resistance. The functionality of 2 ICEs of S. salivarius was investigated. Autonomous conjugative transfer to other S. salivarius strains, to S. thermophilus, and to Enterococcus faecalis was observed. The analysis of the ICE-fda border sequence in these transconjugants allowed the localization of the DNA cutting site of the ICE integrase.IMPORTANCE The ICESt3 subfamily of ICEs appears to be widespread in streptococci and targets diverse chromosomal integration sites. These ICEs carry diverse cargo genes that can confer a selective advantage to the host strain. The maintenance of these mobile genetic elements likely relies in part on self-encoded restriction-modification systems. In this study, intra- and interspecies transfer was demonstrated for 2 ICEs of S. salivarius Closely related ICEs were also detected in silico in other Streptococcus species (S. pneumoniae and S. parasanguinis), thus indicating that diffusion of ICESt3-related elements probably plays a significant role in horizontal gene transfer (HGT) occurring in the oral cavity but also in the digestive tract, where S. salivarius is present.

Resistance Genes and Genetic Elements Associated with Antibiotic Resistance in Clinical and Commensal Isolates of Streptococcus salivarius.

The diversity of clinical (n = 92) and oral and digestive commensal (n = 120) isolates of Streptococcus salivarius was analyzed by multilocus sequence typing (MLST). No clustering of clinical or commensal strains can be observed in the phylogenetic tree. Selected strains (92 clinical and 46 commensal strains) were then examined for their susceptibilities to tetracyclines, macrolides, lincosamides, aminoglycosides, and phenicol antibiotics. The presence of resistance genes tet(M), tet(O), erm(A), erm(B), mef(A/E), and catQ and associated genetic elements was investigated by PCR, as was the genetic linkage of resistance genes. High rates of erythromycin and tetracycline resistance were observed among the strains. Clinical strains displayed either the erm(B) (macrolide-lincosamide-streptogramin B [MLSB] phenotype) or mef(A/E) (M phenotype) resistance determinant, whereas almost all the commensal strains harbored the mef(A/E) resistance gene, carried by a macrolide efflux genetic assembly (MEGA) element. A genetic linkage between a macrolide resistance gene and genes of Tn916 was detected in 23 clinical strains and 5 commensal strains, with a predominance of Tn3872 elements (n = 13), followed by Tn6002 (n = 11) and Tn2009 (n = 4) elements. Four strains harboring a mef(A/E) gene were also resistant to chloramphenicol and carried a catQ gene. Sequencing of the genome of one of these strains revealed that these genes colocalized on an IQ-like element, as already described for other viridans group streptococci. ICESt3-related elements were also detected in half of the isolates. This work highlights the potential role of S. salivarius in the spread of antibiotic resistance genes both in the oral sphere and in the gut.

Detection and Quantification of Conjugative Transfer of Mobile Genetic Elements Carrying Antibiotic Resistance Genes.

Multidrug resistance, due to acquired antimicrobial resistance genes, is increasingly reported in the zoonotic pathogen Streptococcus suis. Most of these resistance genes are carried by chromosomal Mobile Genetic Elements (MGEs), in particular, Integrative and Conjugative Elements (ICEs) and Integrative and Mobilizable Elements (IMEs). ICEs and IMEs frequently form tandems or nested composite elements, which make their identification difficult. To evaluate their mobility, it is necessary to (i) select the suitable donor-recipient pairs for mating assays, (ii) do PCR excision tests to confirm that the genetic element is able to excise from the chromosome as a circular intermediate, and (iii) evaluate the transfer of the genetic element by conjugation by doing mating assays. In addition to a dissemination of resistance genes between S. suis strains, MGEs can lead to a spreading of resistance genes in the environment and toward pathogenic bacteria. This propagation had to be considered in a One Health perspective.

Thermo-regulated adhesion of the Streptococcus thermophilus Δrgg0182 strain.

The physicochemical determinants governing the temperature-dependent adhesion of Streptococcus thermophilus to abiotic surfaces are identified under physiological condition for cells either lacking or not the Rgg0182 transcriptional regulator involved in their thermal adaptation. For that purpose, the wild type LMG18311 strain and Δrgg0182 mutant were imaged using highly resolved atomic force microscopy (AFM) at various cell growth temperatures (42 to 55 °C). The corresponding hydrophobic/hydrophilic balance of the cells was quantitatively addressed via the measurement by chemical force microcopy of their adhesion to a reference hydrophobic surface. Analysis of force-separation distance curves further allowed us to discriminate cell surfaces according to the presence or absence of biopolymers. These results were interpreted in relation to the measured adhesion of the Δrgg0182 mutant onto the hydrophobic wall of microwells in the temperature range from 46 to 52 °C. It is evidenced that the viscoelastic Δrgg0182 cell envelop behaves as a thermo-responsive film whose hydrophobicity increases with increasing temperature, thereby favoring cell attachment to hydrophobic surfaces. Regardless cell growth temperature, wild-type cells do not attach to hydrophobic surfaces and the presence of the Rgg0182 transcriptional regulator is associated with the synthesis of hydrophilic cell surface biopolymers. Throughout, the impact of electrostatics on bioadhesion is ruled out upon examination of electrohydrodynamic cell properties at 50 °C.

Transfer of the Integrative and Conjugative Element ICESt3 of Streptococcus thermophilus in Physiological Conditions Mimicking the Human Digestive Ecosystem.

Metagenome analyses of the human microbiome suggest that horizontal gene transfer (HGT) is frequent in these rich and complex microbial communities. However, so far, only a few HGT studies have been conducted in vivo. In this work, three different systems mimicking the physiological conditions encountered in the human digestive tract were tested, including (i) the TNO gastro-Intestinal tract Model 1 (TIM-1) system (for the upper part of the intestine), (ii) the ARtificial COLon (ARCOL) system (to mimic the colon), and (iii) a mouse model. To increase the likelihood of transfer by conjugation of the integrative and conjugative element studied in the artificial digestive systems, bacteria were entrapped in alginate, agar, and chitosan beads before being placed in the different gut compartments. The number of transconjugants detected decreased, while the complexity of the ecosystem increased (many clones in TIM-1 but only one clone in ARCOL). No clone was obtained in a natural digestive environment (germfree mouse model). In the human gut, the richness and diversity of the bacterial community would offer more opportunities for HGT events to occur. In addition, several factors (SOS-inducing agents, microbiota-derived factors) that potentially increase in vivo HGT efficiency were not tested here. Even if HGT events are rare, expansion of the transconjugant clones can happen if ecological success is fostered by selecting conditions or by events that destabilize the microbial community. IMPORTANCE The human gut microbiota plays a key role in maintaining normal host physiology and health, but its homeostasis is fragile. During their transit in the gastrointestinal tract, bacteria conveyed by food can exchange genes with resident bacteria. New traits acquired by HGT (e.g., new catabolic properties, bacteriocins, antibiotic resistance) can impact the gut microbial composition and metabolic potential. We showed here that TIM-1, a system mimicking the upper digestive tract, is a useful tool to evaluate HGT events in conditions closer to the physiological ones. Another important fact pointed out in this work is that Enterococcus faecalis is a good candidate for foreign gene acquisition. Due to its high ability to colonize the gut and acquire mobile genetic elements, this commensal bacterium could serve as an intermediate for HGT in the human gut.

A New Integrative and Mobilizable Element Is a Major Contributor to Tetracycline Resistance in Streptococcus dysgalactiae subsp. equisimilis.

Tetracycline resistance in streptococci is mainly due to ribosomal protection mediated by the tet(M) gene that is usually located in the integrative and conjugative elements (ICEs) of the Tn916-family. In this study, we analyzed the genes involved in tetracycline resistance and the associated mobile genetic elements (MGEs) in Streptococcus dysgalactiae subsp. equisimilis (SDSE) causing invasive disease. SDSE resistant to tetracycline collected from 2012 to 2019 in a single hospital and from 2018 in three other hospitals were analyzed by whole genome sequencing. Out of a total of 84 SDSE isolates, 24 (28.5%) were resistant to tetracycline due to the presence of tet(M) (n = 22), tet(W) (n = 1), or tet(L) plus tet(W) (n = 1). The tet(M) genes were found in the ICEs of the Tn916-family (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, which were closely related, and all belonged to CC15. In conclusion, tetracycline resistance in SDSE is mostly due to the tet(M) gene associated with ICEs belonging to the Tn916-family and a new IME. This new IME is a major cause of tetracycline resistance in invasive Streptococcus dysgalactiae subsp. equisimilis in our settings.

A new twin expands the VirB8-like protein family.

Conjugative transfer is mediated by specialized type IV secretion systems (T4SSs). However, their architecture and mode of function remain poorly defined in Gram-positives. In this issue of Structure, Jäger et al. reveal an exclusive assembly of PrgL and illustrate the importance of its structural organization in pCF10 conjugative transfer.

FirmiData: a set of 40 genomes of Firmicutes with a curated annotation of ICEs and IMEs.

OBJECTIVES: 'Integrative and Conjugative Elements' (ICEs) and 'Integrative and Mobilizable Elements' (IMEs) are two classes of mobile genetic elements that are complex to detect and delineate. Therefore, they are yet poorly annotated in bacterial genomes. FirmiData provides to the scientific community of microbiologists and bioinformaticians a reference resource of annotated ICEs and of IMEs from Firmicutes. It illustrates their prevalence and their diversity but also gives information on their organization. FirmiData was designed to assist the scientific community in identifying and annotating these elements by using the sequences of these ICEs and IMEs for the identification of related elements in other genomes of Firmicutes. Therefore, Firmidata meets the needs of the scientific community. DATA DESCRIPTION: Firmidata provides a manually curated annotation of 98 ICEs and 148 IMEs identified in 40 chromosomes of Firmicutes. The delineation at the nucleotide level of almost all of these elements allows for the characterization of the genes they carry.

ICEscreen: a tool to detect Firmicute ICEs and IMEs, isolated or enclosed in composite structures.

Mobile Genetic Elements (MGEs) are integrated in bacterial genomes and key elements that drive prokaryote genome evolution. Among them are Integrative and Conjugative Elements (ICEs) and Integrative Mobilizable Elements (IMEs) which are important for bacterial fitness since they frequently carry genes participating in important bacterial adaptation phenotypes such as antibiotic resistance, virulence or specialized metabolic pathways. Although ICEs and IMEs are widespread, they are as yet almost never annotated in public bacterial genomes. To address the need of dedicated strategies for the annotation of these elements, we developed ICEscreen, a tool that introduces two new features to detect ICEs and IMEs in Firmicute genomes. First, ICEscreen uses an efficient strategy to detect Signature Proteins of ICEs and IMEs based on a database dedicated to Firmicutes and composed of manually curated proteins and Hidden Markov Models (HMM) profiles. Second, ICEscreen includes a new original algorithm that detects composite structures of ICEs and IMEs that are frequent in genomes of Firmicutes but are currently not resolved by any other tool. We benchmarked ICEscreen on experimentally supported elements and on a public dataset of 246 manually annotated elements including the genomes of 40 Firmicutes and demonstrate its efficiency to detect ICEs and IMEs.

Prevalence and mobility of integrative and conjugative elements within a Streptomyces natural population.

Horizontal Gene Transfer (HGT) is a powerful force generating genomic diversity in bacterial populations. HGT in Streptomyces is in large part driven by conjugation thanks to plasmids, Integrative and Conjugative elements (ICEs) and Actinomycete ICEs (AICEs). To investigate the impact of ICE and AICE conjugation on Streptomyces genome evolution, we used in silico and experimental approaches on a set of 11 very closely related strains isolated from a millimeter scale rhizosphere population. Through bioinformatic searches of canonical conjugation proteins, we showed that AICEs are the most frequent integrative conjugative elements, with the central chromosome region being a hotspot for integrative element insertion. Strains exhibited great variation in AICE composition consistent with frequent HGT and/or gene loss. We found that single insertion sites can be home to different elements in different strains (accretion) and conversely, elements belonging to the same family can be found at different insertion sites. A wide variety of cargo genes was present in the AICEs with the potential to mediate strain-specific adaptation (e.g., DNA metabolism and resistance genes to antibiotic and phages). However, a large proportion of AICE cargo genes showed hallmarks of pseudogenization, consistent with deleterious effects of cargo genes on fitness. Pock assays enabled the direct visualization of conjugal AICE transfer and demonstrated the transfer of AICEs between some, but not all, of the isolates. Multiple AICEs were shown to be able to transfer during a single mating event. Although we did not obtain experimental evidence for transfer of the sole chromosomal ICE in this population, genotoxic stress mediated its excision from the chromosome, suggesting its functionality. Our results indicate that AICE-mediated HGT in Streptomyces populations is highly dynamic, with likely impact on strain fitness and the ability to adapt to environmental change.

The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation.

BACKGROUND: Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. RESULTS: In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. CONCLUSIONS: These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

Update on the Mechanisms of Antibiotic Resistance and the Mobile Resistome in the Emerging Zoonotic Pathogen Streptococcus suis.

Streptococcus suis is a zoonotic pathogen causing important economic losses in swine production. The most commonly used antibiotics in swine industry are tetracyclines, beta-lactams, and macrolides. Resistance to these antibiotics has already been observed worldwide (reaching high rates for macrolides and tetracyclines) as well as resistance to aminoglycosides, fluoroquinolones, amphenicols, and glycopeptides. Most of the resistance mechanisms are encoded by antibiotic resistance genes, and a large part are carried by mobile genetic elements (MGEs) that can be transferred through horizontal gene transfer. This review provides an update of the resistance genes, their combination in multidrug isolates, and their localization on MGEs in S. suis. It also includes an overview of the contribution of biofilm to antimicrobial resistance in this bacterial species. The identification of resistance genes and study of their localization in S. suis as well as the environmental factors that can modulate their dissemination appear essential in order to decipher the role of this bacterium as a reservoir of antibiotic genes for other species.

Structural and Biochemical Analysis of OrfG: The VirB8-like Component of the Conjugative Type IV Secretion System of ICESt3 From Streptococcus thermophilus.

Conjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural analysis of VirB8-like proteins and discusses the interplay between tertiary and quaternary structures of these proteins as an essential component of the conjugative transfer.

The CHAP domain of Cse functions as an endopeptidase that acts at mature septa to promote Streptococcus thermophilus cell separation.

Cell separation is dependent on cell wall hydrolases that cleave the peptidoglycan shared between daughter cells. In Streptococcus thermophilus, this step is performed by the Cse protein whose depletion resulted in the formation of extremely long chains of cells. Cse, a natural chimeric enzyme created by domain shuffling, carries at least two important domains for its activity: the LysM expected to be responsible for the cell wall-binding and the CHAP domain predicted to contain the active centre. Accordingly, the localization of Cse on S. thermophilus cell surface has been undertaken by immunogold electron and immunofluorescence microscopies using of antibodies raised against the N-terminal end of this protein. Immunolocalization shows the presence of the Cse protein at mature septa. Moreover, the CHAP domain of Cse exhibits a cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis and S. thermophilus. Additionally, RP-HPLC analysis of muropeptides released from B. subtilis and S. thermophilus cell wall after digestion with the CHAP domain shows that Cse is an endopeptidase. Altogether, these results suggest that Cse is a cell wall hydrolase involved in daughter cell separation of S. thermophilus.

Abundance, Diversity and Role of ICEs and IMEs in the Adaptation of Streptococcus salivarius to the Environment.

Streptococcus salivarius is a significant contributor to the human oral, pharyngeal and gut microbiomes that contribute to the maintenance of health. The high genomic diversity observed in this species is mainly caused by horizontal gene transfer. This work aimed to evaluate the contribution of integrative and conjugative elements (ICEs) and integrative and mobilizable elements (IMEs) in S. salivarius genome diversity. For this purpose, we performed an in-depth analysis of 75 genomes of S. salivarius and searched for signature genes of conjugative and mobilizable elements. This analysis led to the retrieval of 69 ICEs, 165 IMEs and many decayed elements showing their high prevalence in S. salivarius genomes. The identification of almost all ICE and IME boundaries allowed the identification of the genes in which these elements are inserted. Furthermore, the exhaustive analysis of the adaptation genes carried by these elements showed that they encode numerous functions such as resistance to stress, to antibiotics or to toxic compounds, and numerous enzymes involved in diverse cellular metabolic pathways. These data support the idea that not only ICEs but also IMEs and decayed elements play an important role in S. salivarius adaptation to the environment.

Mobilization of IMEs Integrated in the oriT of ICEs Involves Their Own Relaxase Belonging to the Rep-Trans Family of Proteins.

Integrative mobilizable elements (IMEs) are widespread but very poorly studied integrated elements that can excise and hijack the transfer apparatus of co-resident conjugative elements to promote their own spreading. Sixty-four putative IMEs, harboring closely related mobilization and recombination modules, were found in 14 Streptococcus species and in Staphylococcus aureus. Fifty-three are integrated into the origin of transfer (oriT) of a host integrative conjugative element (ICE), encoding a MobT relaxase and belonging to three distant families: ICESt3, Tn916, and ICE6013. The others are integrated into an unrelated IME or in chromosomal sites. After labeling by an antibiotic resistance gene, the conjugative transfer of one of these IMEs (named IME_oriTs) and its host ICE was measured. Although the IME is integrated in an ICE, it does not transfer as a part of the host ICE (no cis-mobilization). The IME excises and transfers separately from the ICE (without impacting its transfer rate) using its own relaxase, distantly related to all known MobT relaxases, and integrates in the oriT of the ICE after transfer. Overall, IME_oriTs use MobT-encoding ICEs both as hosts and as helpers for conjugative transfer. As half of them carry lsa(C), they actively participate in the dissemination of lincosamide-streptogramin A-pleuromutilin resistance among Firmicutes.

Chromosomal Conjugative and Mobilizable Elements in Streptococcus suis: Major Actors in the Spreading of Antimicrobial Resistance and Bacteriocin Synthesis Genes.

Streptococcus suis is a zoonotic pathogen suspected to be a reservoir of antimicrobial resistance (AMR) genes. The genomes of 214 strains of 27 serotypes were screened for AMR genes and chromosomal Mobile Genetic Elements (MGEs), in particular Integrative Conjugative Elements (ICEs) and Integrative Mobilizable Elements (IMEs). The functionality of two ICEs that host IMEs carrying AMR genes was investigated by excision tests and conjugation experiments. In silico search revealed 416 ICE-related and 457 IME-related elements. These MGEs exhibit an impressive diversity and plasticity with tandem accretions, integration of ICEs or IMEs inside ICEs and recombination between the elements. All of the detected 393 AMR genes are carried by MGEs. As previously described, ICEs are major vehicles of AMR genes in S. suis. Tn5252-related ICEs also appear to carry bacteriocin clusters. Furthermore, whereas the association of IME-AMR genes has never been described in S. suis, we found that most AMR genes are actually carried by IMEs. The autonomous transfer of an ICE to another bacterial species (Streptococcus thermophilus)-leading to the cis-mobilization of an IME carrying tet(O)-was obtained. These results show that besides ICEs, IMEs likely play a major role in the dissemination of AMR genes in S. suis.