A Genome-Wide CRISPR Interference Screen Reveals an StkP-Mediated Connection between Cell Wall Integrity and Competence in Streptococcus salivarius.

Competence is one of the most efficient bacterial evolutionary and adaptative strategies by synchronizing production of antibacterial compounds and integration of DNA released by dead cells. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing a short time window of activation in which only part of the population is responsive. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. However, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using CRISPR interference and performed three complementary screens to investigate competence genetic regulation in the human commensal Streptococcus salivarius. We show that initiation of competence increases upon cell wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report a key role for StkP, a serine-threonine kinase known to regulate cell wall homeostasis. We show that StkP controls competence by a mechanism that reacts to peptidoglycan fragments. Together, our data suggest a key cell wall sensing mechanism coupling competence to cell envelope integrity. IMPORTANCE Survival of human commensal streptococci in the digestive tract requires efficient strategies which must be tightly and collectively controlled for responding to competitive pressure and drastic environmental changes. In this context, the autocrine signaling system ComRS controlling competence for natural transformation and predation in salivarius streptococci could be seen as a multi-input device integrating a variety of environmental stimuli. In this work, we revealed novel positive and negative competence modulators by using a genome-wide CRISPR interference strategy. Notably, we highlighted an unexpected connection between bacterial envelope integrity and competence activation that involves several cell wall sensors. Together, these results showcase how commensal streptococci can fine-tune the pheromone-based competence system by responding to multiple inputs affecting their physiological status in order to calibrate an appropriate collective behavior.

Gastric Non-Helicobacter pylori Urease-Positive Staphylococcus epidermidis and Streptococcus salivarius Isolated from Humans Have Contrasting Effects on H. pylori-Associated Gastric Pathology and Host Immune Responses in a Murine Model of Gastric Cancer.

In populations with similar prevalence of Helicobacter pylori infection, cancer risk can vary dramatically. Changes in composition or structure of bacterial communities in the stomach, either at the time of exposure or over the course of H. pylori infection, may contribute to gastric pathology. In this study, a population of 37 patients from the low-gastric-cancer-risk (LGCR) region of Tumaco, Colombia, and the high-gastric-cancer-risk (HGCR) region of Túquerres, Colombia, were recruited for gastric endoscopy. Antral biopsy specimens were processed for histology and bacterial isolation. Fifty-nine distinct species among 26 genera were isolated by aerobic, anaerobic, and microaerobic culture and confirmed by 16S rRNA analysis. Urease-positive Staphylococcus epidermidis and Streptococcus salivarius were frequently isolated from gastric biopsy specimens. We asked whether coinfection of H. pylori with urease-positive S. salivarius and/or S. epidermidis had a demonstrable effect on H. pylori-induced gastritis in the germfree (GF) INS-GAS mouse model. Coinfections with S. salivarius and/or S. epidermidis did not affect gastric H. pylori colonization. At 5 months postinfection, GF INS-GAS mice coinfected with H. pylori and S. salivarius had statistically higher pathological scores in the stomachs than mice infected with H. pylori only or H. pylori with S. epidermidis (P < 0.05). S. epidermidis coinfection with H. pylori did not significantly change stomach pathology, but levels of the proinflammatory cytokine genes Il-1ß, Il-17A , and Il-22 were significantly lower than in H. pylori-monoinfected mice. This study demonstrates that non-H. pylori urease-positive bacteria may play a role in the severity of H. pylori-induced gastric cancer in humans. IMPORTANCE Chronic infection with H. pylori is the main cause of gastric cancer, which is a global health problem. In two Colombian populations with high levels of H. pylori prevalence, the regional gastric cancer rates are considerably different. Host genetic background, H. pylori biotype, environmental toxins, and dietary choices are among the known risk factors for stomach cancer. The potential role of non-H. pylori gastric microbiota in gastric carcinogenesis is being increasingly recognized. In this study, we isolated 59 bacterial species from 37 stomach biopsy samples of Colombian patients from both low-gastric-cancer-risk and high-gastric-cancer-risk regions. Urease-positive S. epidermidis and S. salivarius commonly cultured from the stomachs, along with H. pylori, were inoculated into germfree INS-GAS mice. S. salivarius coinfection with H. pylori induced significantly higher gastric pathology than in H. pylori-monoinfected mice, whereas S. epidermidis coinfection caused significantly lower H. pylori-induced proinflammatory cytokine responses than in H. pylori-monoinfected mice. This study reinforces the argument that the non-H. pylori stomach microflora play a role in the severity of H. pylori-induced gastric cancer.

Probiotic Streptococcus salivarius K12 Alleviates Radiation-Induced Oral Mucositis in Mice.

Background: Oral mucositis is the most common oral complication of cancer patients receiving radiotherapy and/or chemotherapy, leading to poor quality of life. Limitations of the current interventions on radiation-induced oral mucositis (RIOM) urge the development of novel therapeutics. Here, we evaluated the treatment outcome of probiotic Streptococcus salivarius K12 on RIOM mice, and oral microbiota that is associated with the progress of RIOM was further investigated. Methods: An experimental RIOM mouse model was established, and S. salivarius K12 was applied to the mouse oral cavity daily. Histological analyses were performed to evaluate the severity of oral mucositis and the treatment outcome of S. salivarius K12. The oral microbiota of mice was further analyzed by 16S rRNA sequencing, microbial culture and qPCR. Results: Irradiation induced conspicuous mucositis in the oral cavity of mice. S. salivarius K12 treatment was beneficial for the healing of RIOM, as reflected by reduced ulcer size, increased basal layer epithelial cellularity and mucosal thickness, and elevated epithelial proliferation and attenuated apoptosis. RIOM mice presented significant oral microbial dysbiosis, with an overgrowth of oral anaerobes. S. salivarius K12 treatment reconstituted the oral microbiota and decreased the abundance of oral anaerobes of RIOM mice. In addition, S. salivarius K12 treatment inhibited NI1060 in Pasteurella genus and downregulated the expression of nitrate reductase. Conclusions: S. salivarius K12 treatment can alleviate RIOM and reconstituted the dysbiotic oral microbiota in mice. S. salivarius K12 may represent a promising adjuvant treatment to improve the quality of life of cancer patients receiving radiotherapy.

Safety assessment of Streptococcus salivarius DB-B5 as a probiotic candidate for oral health.

Streptococcus salivarius DB-B5 was previously isolated from the supragingival plaque of a healthy female adult and selected for development as a probiotic candidate for oral health. Probiotics are an important emerging therapeutic method for preventing, treating, and maintaining oral health. Although S. salivarius is a predominant member of the commensal oral microbiota and generally regarded as a safe species, it is recognized that each strain needs to be comprehensively assessed for safety. This study describes the in silico, in vitro, and clinical testing that were conducted to evaluate the safety of S. salivarius DB-B5. Both 16S rRNA and multi-gene phylogenetic reconstruction was used to confirm the taxonomic identity of this strain. Bioinformatic analysis of the genome demonstrated the absence of transmissible antibiotic resistance genes or virulence factors. Phenotypic testing further showed S. salivarius DB-B5 to be susceptible to clinically relevant antibiotics. S. salivarius DB-B5 displayed weak alpha-hemolysis, and does not produce biogenic amines. In a randomized, double-blind, placebo-controlled clinical study, consumption of S. salivarius DB-B5 at 10 billion CFU/day for 4 weeks by healthy adults was safe and well-tolerated (ClinicalTrials.gov registry number NCT04492631). This work has indicated that S. salivarius DB-B5 is a safe probiotic candidate.

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.

Cationic hydrogels prepared from regioselectively azidated (1→3)-α-d-glucan via crosslinking and amination: Physical and adsorption properties.

Cationic hydrogels with amino groups were successfully prepared using (1→3)-α-d-glucan synthesized by glucosyltransferase J (GtfJ) cloned from Streptococcus salivarius through a three-step reaction: (i) Azido groups were regioselectively introduced at the C6 position of (1→3)-α-d-glucan by a bromination-azidation process (degree of substitution 0.94), (ii) Azido groups were partially crosslinked with 1,8-nonadiyne via a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, (iii) Azido groups that were unused for crosslinking were reduced to amino groups by sodium borohydride (NaBH4). The introduction of amino groups was confirmed quantitatively and qualitatively by elemental, Fourier transform infrared (FT-IR), and nuclear magnetic resonance (NMR) analyses. These cationic hydrogels showed a specific adsorption ability for bovine serum albumin (BSA) over a wide pH range of 4.5-8.0 due to their high pH values at the point of zero charge (pHpzc 8.80-8.92).

Enzymatic polymerization derived engineered polysaccharides as reinforcing fillers of ethylene vinyl acetate composites.

A novel monomer based, controlled enzymatic polymerization was employed to produce an engineered alpha-1,3 glucan polysaccharide. The structure and material properties of the engineered polysaccharide were characterized using various techniques. The use of such engineered polysaccharide as a reinforcing filler of polymers was evaluated using model polymers. For this, the alpha-1,3 glucan was incorporated into ethylene vinyl acetate co-polymer (EVA) matrices with vinyl acetate content of 32% and 40% via a melt processing fabrication process. Various mechanical and rheological properties of the fabricated composites were evaluated. The effect of vinyl acetate content of the EVA resin on the interaction with alpha-1,3 glucan that result in various performances attributes was also investigated and reported. The incorporation of alpha-1,3 glucan in these EVA composites resulted in the improvement of key composite properties, such as toughness, modulus, wear resistance, and hardness showing the reinforcing potential of these engineered polysaccharides.

A TaqMan™-based quantitative PCR screening assay for the probiotic Streptococcus salivarius K12 based on the specific detection of its megaplasmid-associated salivaricin B locus.

In order to assess the colonization efficacy of the oral probiotic Streptococcus salivarius K12, a rapid method for specific detection and enumeration of the strain was developed. Here, we describe a two-step TaqMan™ quantitative PCR assay using primer-probe combinations targeting genes of the locus encoding the lantibiotic bacteriocin salivaricin B.

Application of DNA repair for Streptococcus salivarius DNA-based identification of saliva from ultraviolet-exposed samples.

Forensic samples are commonly influenced by various environmental factors, including ultraviolet (UV) irradiation; thus, forensic applications of DNA repair (e.g., PreCR™, Restorase®) have been investigated, focusing on short tandem repeat typing. However, current DNA-based examinations are used for both human and body fluid identification. This study thus aims to clarify the efficacy of a DNA repair approach for Streptococcus salivarius DNA-based identification of saliva from UV-damaged samples. Artificial UV-damaged genomic DNA of S. salivarius, drop saliva stains, and buccal swabs were used to evaluate the effects of DNA repair on S. salivarius DNA detection by using PreCR™ repair reagent. To evaluate forensic applications, we prepared mock forensic samples by exposing them to environmental conditions. Melting curve analysis following real-time PCR was applied for qualitatively detecting S. salivarius DNA with a specific melting peak of 80.5°C±0.4°C (n=10, mean ± 3SD). Single PCR was used for quantitative and qualitative analyses, whereas dual PCR was used for S. salivarius DNA qualitative detection. DNA repair experiments using artificial UV-damaged samples revealed a significant increase of only the quantitative value of genomic DNA samples by DNA repair. Moreover, significant quantitative DNA repair effects were not observed in all mock forensic samples, indicating the limitations of DNA repair for actual cell-derived DNA samples. Whereas, differences of qualitative results (with or without detection) were generated for mock forensic samples; thus, we consider the DNA repair strategy as an additional approach for S. salivarius DNA-based identification of saliva from environmentally damaged evidence.

Subtle selectivity in a pheromone sensor triumvirate desynchronizes competence and predation in a human gut commensal.

Constantly surrounded by kin or alien organisms in nature, eukaryotes and prokaryotes developed various communication systems to coordinate adaptive multi-entity behavior. In complex and overcrowded environments, they require to discriminate relevant signals in a myriad of pheromones to execute appropriate responses. In the human gut commensal Streptococcus salivarius, the cytoplasmic Rgg/RNPP regulator ComR couples competence to bacteriocin-mediated predation. Here, we describe a paralogous sensor duo, ScuR and SarF, which circumvents ComR in order to disconnect these two physiological processes. We highlighted the recurring role of Rgg/RNPP in the production of antimicrobials and designed a robust genetic screen to unveil potent/optimized peptide pheromones. Further mutational and biochemical analyses dissected the modifiable selectivity toward their pheromone and operating sequences at the subtle molecular level. Additionally, our results highlight how we might mobilize antimicrobial molecules while silencing competence in endogenous populations of human microflora and temper gut disorders provoked by bacterial pathogens.

Streptococcus Salivarius: A Potential Salivary Biomarker for Orofacial Granulomatosis and Crohn's Disease?

BACKGROUND: Orofacial granulomatosis (OFG) is a rare disease characterised by chronic, noncaseating, granulomatous inflammation primarily affecting the oral cavity. Histologically, it is similar to Crohn's disease (CD), and a proportion of patients have both OFG and CD. The cause of OFG remains elusive, but it has been suggested that microbial interactions may be involved. The aim of this study was to compare the salivary microbial composition of subjects with OFG and/or CD and healthy controls. METHODS: Two hundred sixty-one subjects were recruited, of whom 78 had OFG only, 40 had both OFG and CD, 97 had CD only with no oral symptoms, and 46 were healthy controls. Bacterial community profiles were obtained by sequencing the V1-V3 region of the 16S rRNA gene. RESULTS: There were no differences in richness or diversity of the salivary bacterial communities between patient groups and controls. The relative abundance of the Streptococcus salivarius group was raised in patients with OFG or CD only compared with controls, whereas that of the Streptococcus mitis group was lower in CD compared with both OFG and controls. One S. salivarius oligotype made the major contribution to the increased proportions seen in patients with OFG and CD. CONCLUSIONS: The salivary microbiome of individuals with OFG and CD was similar to that found in health, although the proportions of S. salivarius, a common oral Streptococcus, were raised. One specific strain-level oligotype was found to be primarily responsible for the increased levels seen.

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.

Circuitry Rewiring Directly Couples Competence to Predation in the Gut Dweller Streptococcus salivarius.

Small distortions in transcriptional networks might lead to drastic phenotypical changes, especially in cellular developmental programs such as competence for natural transformation. Here, we report a pervasive circuitry rewiring for competence and predation interplay in commensal streptococci. Canonically, in streptococci paradigms such as Streptococcus pneumoniae and Streptococcus mutans, the pheromone-based two-component system BlpRH is a central node that orchestrates the production of antimicrobial compounds (bacteriocins) and incorporates signal from the competence activation cascade. However, the human commensal Streptococcus salivarius does not contain a functional BlpRH pair, while the competence signaling system ComRS directly couples bacteriocin production and competence commitment. This network shortcut might underlie an optimal adaptation against microbial competitors and explain the high prevalence of S. salivarius in the human digestive tract. Moreover, the broad spectrum of bacteriocin activity against pathogenic bacteria showcases the commensal and genetically tractable S. salivarius species as a user-friendly model for competence and bacterial predation.

Simple and rapid identification of saliva by detection of oral streptococci using direct polymerase chain reaction combined with an immunochromatographic strip.

In this paper, we describe the development of a novel method to detect oral bacteria by combining direct polymerase chain reaction (direct PCR) with an immunochromatographic strip (ICS), enabling the identification of saliva in forensic samples. Direct PCR was first used to directly amplify specific oral bacterial sequences (from Streptococcus sanguinis and Streptococcus salivarius) from swab samples, circumventing the need for tedious sample preparation steps such as cell lysis and DNA extraction and purification. The resultant amplicons were then colorimetrically detected on an ICS, a much more convenient, cost-effective, and user-friendly detection method than those currently available, thereby allowing the presence or absence of the target oral bacteria to be determined with the naked eye. Moreover, the entire analysis process was performed rapidly and with ease using this combination of direct PCR amplification from swab samples and ICS-based amplicon detection. This method successfully detected S. sanguinis and S. salivarius in most of the saliva swab samples tested, and returned negative results using blood, semen, urine, and vaginal fluid swab samples. Furthermore, S. sanguinis and S. salivarius were detected in a large number of mock forensic samples using this technique, which suggests that direct PCR and ICS-based detection of oral bacteria is sufficient to demonstrate the presence of saliva. Thus, we believe that the proposed method could be very useful for the identification of saliva in forensic applications.

Genetic organization of Streptococcus salivarius 24SMBc blp-like bacteriocin locus.

In this paper, we describe, for the first time, the genetic organization of the blpU-like cassette in Streptococcus salivarius24SMBc by entire genome sequencing. This strain has recently been found useful and widely applied as an oral probiotic in the prevention of recurrent otitis media. The 24SMBc blpU-like cassette  is 8,023 bp in length, organized in 11 orfs,of which orf8 encodes for the pore-forming peptide bacteriocin, belonging to class IIc, with a double-glycine leader peptide. The first characterization of blplocus was described inStreptococcus pneumoniae, showing a crucial role in interspecies competition within the nasopharynx. The salivarius blpU-like cassette is inserted upstream of the pepX gene in the chromosome. A hypervariable region between pepXand orf1 was found and used as a specific target able to distinguishS. salivarius 24SMBc from all other streptococci. All orfscarried by the blp-like cassette are functionally expressed (qPCR assays). Our results contribute to elucidate the microbial interactions in the nasopharynx, underlining the potential role of  the blp locus in human nasopharyngeal colonization.

Three glycosylated serine-rich repeat proteins play a pivotal role in adhesion and colonization of the pioneer commensal bacterium, Streptococcus salivarius.

Bacterial adhesion is a critical step for colonization of the host. The pioneer colonizer and commensal bacterium of the human gastrointestinal tract, Streptococcus salivarius, has strong adhesive properties but the molecular determinants of this adhesion remain uncharacterized. Serine-rich repeat (SRR) glycoproteins are a family of adhesins that fulfil an important role in adhesion. In general, Gram-positive bacterial genomes have a unique SRR glycoprotein-encoding gene. We demonstrate that S. salivarius expresses three large and glycosylated surface-exposed proteins - SrpA, SrpB and SrpC - that show characteristics of SRR glycoproteins and are secreted through the accessory SecA2/Y2 system. Two glycosyltransferases - GtfE/F - encoded outside of the secA2/Y2 locus, unusually, perform the first step of the sequential glycosylation process, which is crucial for SRR activity. We show that SrpB and SrpC play complementary adhesive roles involved in several steps of the colonization process: auto-aggregation, biofilm formation and adhesion to a variety of host epithelial cells and components. We also show that at least one of the S. salivarius SRR glycoproteins is important for colonization in mice. SrpA, SrpB and SrpC are the main factors underlying the multifaceted adhesion of S. salivarius and, therefore, play a major role in host colonization.

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.

Influence of Oral Probiotic Streptococcus salivarius K12 on Ear and Oral Cavity Health in Humans: Systematic Review.

Traditionally, probiotics are linked to the good health of the intestine and most clinical studies focus on that field. Evidence of oral probiotic use for ear and oral cavity disease prevention with impact on human health is limited. This work reviews existing studies and literature on Streptococcus salivarius K12 as an oral probiotic and effects of S. salivarius K12 on human ear and oral cavity human health. The studies were accessed via database searches: MEDLINE, PubMed, and Elsevier. The search included/focused on/encompassed publications from 2003 to 2016 with keywords related to K12 Streptococcus salivarius, bacteriocin-like inhibitory substances (BLIS) K12, probiotic K12 salivarius, and K12 probiotic health effects. Only a small amount of studies was identified: the total of 68 studies was identified, 35 of which were relevant after screening, and 9 were included in the final analysis. Very little literature is available about the association/correlation between/connection/interrelation of S. salivarius K12 with/and human ear and oral cavity health. S. salivarius K12 may have a role in reducing the occurrence and/or severity of secretory otitis media (SOM) and also in prevention of streptococcal and viral pharyngotonsillitis in children. Research highlights that S. salivarius K12 has shown promising results in treatment of halitosis, but data are still deficient. Further studies need to be initiated to improve understanding of the association of oral probiotic S. salivarius K12 with human ear and oral cavity health.

YMC-2011, a Temperate Phage of Streptococcus salivarius 57.I.

Streptococcus salivarius is an abundant isolate of the oral cavity. The genome of S. salivarius 57.I consists of a 2-Mb chromosome and a 40,758-bp circular molecule, designated YMC-2011. Annotation of YMC-2011 revealed 55 open reading frames, most of them associated with phage production, although plaque formation is not observed in S. salivarius 57.I after lytic induction using mitomycin C. Results from Southern hybridization and quantitative real-time PCR confirmed that YMC-2011 exists extrachromosomally, with an estimated copy number of 3 to 4. Phage particles were isolated from the supernatant of mitomycin C-treated S. salivarius 57.I cultures, and transmission electron microscopic examination indicated that YMC-2011 belongs to the Siphoviridae family. Phylogenetic analysis suggests that phage YMC-2011 and the cos-type phages of Streptococcus thermophilus originated from a common ancestor. An extended -10 element (p L ) and a σ70-like promoter (p R ) were mapped 5' to Ssal_phage00013 (encoding a CI-like repressor) and Ssal_phage00014 (encoding a hypothetical protein), respectively, using 5' rapid amplification of cDNA ends, indicating that YMC-2011 transcribes at least two mRNAs in opposite orientations. Studies using promoter-chloramphenicol acetyltransferase reporter gene fusions revealed that p R , but not p L , was sensitive to mitomycin C induction, suggesting that the switch from lysogenic growth to lytic growth was controlled mainly by the activity of these two promoters. In conclusion, a lysogenic state is maintained in S. salivarius 57.I, presumably by the repression of genes encoding proteins for lytic growth.IMPORTANCE The movement of mobile genetic elements such as bacteriophages and the establishment of lysogens may have profound effects on the balance of microbial ecology where lysogenic bacteria reside. The discovery of phage YMC-2011 from Streptococcus salivarius 57.I suggests that YMC-2011 and Streptococcus thermophilus-infecting phages share an ancestor. Although S. salivarius and S. thermophilus are close phylogenetically, S. salivarius is a natural inhabitant of the human mouth, whereas S. thermophilus is commonly found in the mammary mucosa of bovine species. Thus, the identification of YMC-2011 suggests that horizontal gene transfer via phage infection could take place between species from different ecological niches.

Oral cavities of healthy infants harbour high proportions of Streptococcus salivarius strains with phenotypic and genotypic resistance to multiple classes of antibiotics.

Emerging antibiotic resistance in the oropharyngeal microbiota, of which Streptococcus salivarius is a prominent species, represents a challenge for treating paediatric populations. In this study, we investigated the role of Streptococcussalivarius as a reservoir for antibiotic resistance genes (ARG) in the oral microbiota by analysing 95 Streptococcussalivarius isolates from 22 healthy infants (2-16 months of age). MICs of penicillin G, amoxicillin, erythromycin, tetracycline, doxycycline and streptomycin were determined. ARG profiles were assessed in a subset of 21 strains by next-generation sequencing of genomes, followed by searches of assembled reads against the Comprehensive Antibiotic Resistance Database. Strains resistant to erythromycin, penicillins and tetracyclines were isolated from 83.3, 33.3 and 16.6 %, respectively, of infants aged 2 to 8 months with no prior antibiotic treatment. These percentages were100.0, 66.6 and 50.0 %, by 13 to 16 months of age. ARG or polymorphisms associated with antibiotic resistance were the most prevalent and involved genes for macrolide efflux (mel, mefA/E and macB), ribosomal protection [erm(B), tet(M) and tet(O)] and ß-lactamase-like proteins. Phylogenetically related strains showing multidrug-resistant phenotypes harboured multidrug efflux ARG. Polymorphic genes associated with antibiotic resistance to drugs affecting DNA replication, folate synthesis, RNA/protein synthesis and regulators of antibiotic stress responses were detected. These data imply that Streptococcussalivarius strains established during maturation of the oral microbiota harbour a diverse array of functional ARG, even in the absence of antibiotic selective pressures, highlighting a potential role for this species in shaping antibiotic susceptibility profiles of oropharyngeal communities.