Biofilm and bacterial membrane vesicles: recent advances.

INTRODUCTION: Bacterial Membrane Vesicles (MVs) play important roles in cell-to-cell communication and transport of several molecules. Such structures are essential components of Extracellular Polymeric Substances (EPS) biofilm matrix of many bacterial species displaying a structural function and a role in virulence and pathogenesis. AREAS COVERED: In this review were included original articles from the last ten years by searching the keywords 'biofilm' and 'vesicles' on PUBMED and Scopus databases. The articles available in literature mainly describe a positive correlation between bacterial MVs and biofilms formation. The research on Espacenet and Google Patent databases underlines the available patents related to the application of both biofilm MVs and planktonic MVs in inhibiting biofilm formation. EXPERT OPINION: This review covers and analyzes recent advances in the study of the relationship between bacterial vesicles and biofilm. The huge number of papers discussing the role of MVs confirms the interest aimed at developing new applications in the medical field. The study of the MVs composition and biogenesis may contribute to the identification of components which could be (i) the target for the development of new drugs inhibiting the biofilm establishment; (ii) candidates for the development of vaccines; (iii) biomarkers for the diagnosis of bacterial infections.

Interactions of Gram-Positive Bacterial Membrane Vesicles and Hosts: Updates and Future Directions.

Extracellular vesicles (EVs) are lipid bilayers derived from cell membranes, released by both eukaryotic cells and bacteria into the extracellular environment. During production, EVs carry proteins, nucleic acids, and various compounds, which are then released. While Gram-positive bacteria were traditionally thought incapable of producing EVs due to their thick peptidoglycan cell walls, recent studies on membrane vesicles (MVs) in Gram-positive bacteria have revealed their significant role in bacterial physiology and disease progression. This review explores the current understanding of MVs in Gram-positive bacteria, including the characterization of their content and functions, as well as their interactions with host and bacterial cells. It offers a fresh perspective to enhance our comprehension of Gram-positive bacterial EVs.

Lactobacillus rhamnosus GG cell-free supernatant as a novel anti-cancer adjuvant.

BACKGROUND: Gut microbiota modulation has been demonstrated to be effective in protecting patients against detrimental effects of anti-cancer therapies, as well as to improve the efficacy of certain anti-cancer treatments. Among the most characterized probiotics, Lactobacillus rhamnosus GG (LGG) is currently utilized in clinics to alleviate diarrhea, mucositis or intestinal damage which might be associated with several triggers, including Clostridium difficile infections, inflammatory gut diseases, antibiotic consumption, chemotherapy or radiation therapy. Here, we investigate whether LGG cell-free supernatant (LGG-SN) might exert anti-proliferative activity toward colon cancer and metastatic melanoma cells. Moreover, we assess the potential adjuvant effect of LGG-SN in combination with anti-cancer drugs. METHODS: LGG-SN alone or in combination with either 5-Fuorouracil and Irinotecan was used to treat human colon and human melanoma cancer cell lines. Dimethylimidazol-diphenyl tetrazolium bromide assay was employed to detect cellular viability. Trypan blue staining, anti-cleaved caspase-3 and anti-total versus anti-cleaved PARP western blots, and annexin V/propidium iodide flow cytometry analyses were used to assess cell death. Flow cytometry measurement of cellular DNA content (with propidium iodide staining) together with qPCR analysis of cyclins expression were used to assess cell cycle. RESULTS: We demonstrate that LGG-SN is able to selectively reduce the viability of cancer cells in a concentration-dependent way. While LGG-SN does not exert any anti-proliferative activity on control fibroblasts. In cancer cells, the reduction in viability is not associated with apoptosis induction, but with a mitotic arrest in the G2/M phase of cell cycle. Additionally, LGG-SN sensitizes cancer cells to both 5-Fluorouracil and Irinotecan, thereby showing a positive synergistic action. CONCLUSION: Overall, our results suggest that LGG-SN may contain one or more bioactive molecules with anti-cancer activity which sensitize cancer cells to chemotherapeutic drugs. Thus, LGG could be proposed as an ideal candidate for ground-breaking integrated approaches to be employed in oncology, to reduce chemotherapy-related side effects and overcome resistance or relapse issues, thus ameliorating the therapeutic response in cancer patients.

Streptococcus salivarius 24SMBc Genome Analysis Reveals New Biosynthetic Gene Clusters Involved in Antimicrobial Effects on Streptococcus pneumoniae and Streptococcus pyogenes.

Streptococcus salivarius 24SMBc is an oral probiotic with antimicrobial activity against the otopathogens Streptococcus pyogenes and Streptococcus pneumoniae. Clinical studies have reinforced its role in reducing the recurrence of upper respiratory tract infections (URTIs) and rebalancing the nasal microbiota. In this study, for the first time, we characterized 24SMBc by whole genome sequencing and annotation; likewise, its antagonistic activity vs. Streptococcus pneumoniae and Streptococcus pyogenes was evaluated by in vitro co-aggregation and competitive adherence tests. The genome of 24SMBc comprises 2,131,204 bps with 1933 coding sequences (CDS), 44 tRNA, and six rRNA genes and it is categorized in 319 metabolic subsystems. Genome mining by BAGEL and antiSMASH tools predicted three novel biosynthetic gene clusters (BGCs): (i) a Blp class-IIc bacteriocin biosynthetic cluster, identifying two bacteriocins blpU and blpK; (ii) an ABC-type bacteriocin transporter; and (iii) a Type 3PKS (Polyketide synthase) involved in the mevalonate pathway for the isoprenoid biosynthetic process. Further analyses detected two additional genes for class-IIb bacteriocins and 24 putative adhesins and aggregation factors. Finally, in vitro assays of 24SMBc showed significant anti-adhesion and co-aggregation effects against Streptococcus pneumoniae strains, whereas it did not act as strongly against Streptococcus pyogenes. In conclusion, we identified a novel blpU-K bacteriocin-encoding BGC and two class-IIb bacteriocins involved in the activity against Streptococcus pneumoniae and Streptococcus pyogenes; likewise the type 3PKS pathway could have beneficial effects for the host including antimicrobial activity. Furthermore, the presence of adhesins and aggregation factors might be involved in the marked in vitro activity of co-aggregation with pathogens and competitive adherence, showing an additional antibacterial activity not solely related to metabolite production. These findings corroborate the antimicrobial activity of 24SMBc, especially against Streptococcus pneumoniae belonging to different serotypes, and further consolidate the use of this strain in URTIs in clinical settings.

Microbiome differences in periodontal, peri-implant, and healthy sites: a cross-sectional pilot study.

OBJECTIVES: To explore microbial communities associated with health and disease status around teeth and dental implants. MATERIALS AND METHODS: A total of 10 healthy, 24 periodontitis, and 24 peri-implant sites from 24 patients were sequenced by next-generation sequencing. Microbial DNA was extracted and 16S rRNA gene was amplified. Bioinformatic analyses were performed using quantitative insights into microbial ecology (QIIME), linear discriminant analysis effect size (LEfSE), and STAMP. RESULTS: Differences in microbial diversity across three types of sites were not statistically significant. Several genera and species were more prevalent in healthy compared with diseased sites, including Lautropia, Rothia and Capnocytophaga and Kingella. Among diseased sites, Peptostreptococcaceae, Dialister, Mongibacterium, Atopobium, and Filifactor were over-represented in peri-implantitis sites, while Bacteroidales was more abundant in periodontitis sites. CONCLUSIONS: Diseased periodontal and peri-implant sites and corresponding healthy sites have distinct microbiological profiles. These findings suggest that microbial analyses could identify biomarkers for periodontal health and disease and lead to the development of new strategies to improve periodontal health and treat peri-implant and periodontal diseases. CLINICAL RELEVANCE: The study contributes to improving our understanding of healthy, periodontally affected, and peri-implantitis sites which can improve our ability to diagnose, monitor, and manage these oral conditions.

Antimicrobial properties of Lactobacillus cell-free supernatants against multidrug-resistant urogenital pathogens.

The healthy vaginal microbiota is dominated by Lactobacillus spp., which provide an important critical line of defense against pathogens, as well as giving beneficial effects to the host. We characterized L. gasseri 1A-TV, L. fermentum 18A-TV, and L. crispatus 35A-TV, from the vaginal microbiota of healthy premenopausal women, for their potential probiotic activities. The antimicrobial effects of the 3 strains and their combination against clinical urogenital bacteria were evaluated together with the activities of their metabolites produced by cell-free supernatants (CFSs). Their beneficial properties in terms of ability to interfere with vaginal pathogens (co-aggregation, adhesion to HeLa cells, biofilm formation) and antimicrobial activity mediated by CFSs were assessed against multidrug urogenital pathogens (S. agalactiae, E. coli, KPC-producing K. pneumoniae, S. aureus, E. faecium VRE, E. faecalis, P. aeruginosa, P. mirabilis, P. vulgaris, C. albicans, C. glabrata). The Lactobacilli tested exhibited an extraordinary ability to interfere and co-aggregate with urogenital pathogens, except for Candida spp., as well as to adhere to HeLa cells and to produce biofilm in the Lactobacillus combination. Lactobacillus CFSs and their combination revealed a strong bactericidal effect on the multidrug resistant indicator strains tested, except for E. faecium and E. faecalis. The antimicrobial activity was maintained after heat treatment but decreased after enzymatic treatment. All Lactobacilli showed lactic dehydrogenase activity and production of D- and L-lactic acid isomers on Lactobacillus CFSs, while only 1A-TV and 35A-TV released hydrogen peroxide and carried helveticin J and acidocin A bacteriocins. These results suggest that they can be employed as a new vaginal probiotic formulation and bio-therapeutic preparation against urogenital infections. Further, in vivo studies are needed to evaluate human health benefits in clinical situations.

Potential Associations Among Alteration of Salivary miRNAs, Saliva Microbiome Structure, and Cognitive Impairments in Autistic Children.

Recent evidence has demonstrated that salivary molecules, as well as bacterial populations, can be perturbed by several pathological conditions, including neuro-psychiatric diseases. This relationship between brain functionality and saliva composition could be exploited to unveil new pathological mechanisms of elusive diseases, such as Autistic Spectrum Disorder (ASD). We performed a combined approach of miRNA expression profiling by NanoString technology, followed by validation experiments in qPCR, and 16S rRNA microbiome analysis on saliva from 53 ASD and 27 neurologically unaffected control (NUC) children. MiR-29a-3p and miR-141-3p were upregulated, while miR-16-5p, let-7b-5p, and miR-451a were downregulated in ASD compared to NUCs. Microbiome analysis on the same subjects revealed that Rothia, Filifactor, Actinobacillus, Weeksellaceae, Ralstonia, Pasteurellaceae, and Aggregatibacter increased their abundance in ASD patients, while Tannerella, Moryella and TM7-3 decreased. Variations of both miRNAs and microbes were statistically associated to different neuropsychological scores related to anomalies in social interaction and communication. Among miRNA/bacteria associations, the most relevant was the negative correlation between salivary miR-141-3p expression and Tannerella abundance. MiRNA and microbiome dysregulations found in the saliva of ASD children are potentially associated with cognitive impairments of the subjects. Furthermore, a potential cross-talking between circulating miRNAs and resident bacteria could occur in saliva of ASD.

Bacteriotherapy with Streptococcus salivarius 24SMB and Streptococcus oralis 89a oral spray for children with recurrent streptococcal pharyngotonsillitis: a randomized placebo-controlled clinical study.

PURPOSE: Group A beta-hemolytic Streptococcus (GABHS) causes a recurrent acute pharyngotonsillitis (RAPT) in children. Moreover, the repeated use of antibiotics contributes to its resistance. However, S. Salivarius 24SMB and S. oralis 89a were effective probiotics in other infections. Thus, we decided to evaluate this combination efficacy compared to placebo in RAPT. METHODS: Patients with microbiologically confirmed GABHS were enrolled in this randomized, placebo-controlled trial. They received the aforementioned combination or placebo as an oral spray. We investigated episodes of frequency and duration, need for antibiotics, school days lost, the treatment impact on life quality, treatment compliance and side effects during a 90-day treatment and a 6-month follow-up. RESULTS: We included 41 patients in each group. The mean number of GABHS infection was significantly lower during both study periods for the two groups. However, our treatment group showed a lower rate. Moreover, the probiotic group had a lower mean number and a shorter median duration of GABHS episodes during both study periods than controls. Furthermore, the mean duration of antibiotic treatment was lower in the probiotic group during the 90-day and 6-month follow-up periods. Similarly, patients in the probiotic group showed a significantly lower mean number of absence days from school but higher EQ-VAS score. Indeed, all patients included were compliant to treatment. CONCLUSIONS: We identified potential probiotics, possessing desirable features against GABHS pharyngotonsillitis. Our findings represent the first evidence which throws the light on using these probiotics that can reduce antibiotics use which did not have efficient results regarding recurrence.

Gut Microbiota and Cancer: From Pathogenesis to Therapy.

Cancer is a multifactorial pathology and it represents the second leading cause of death worldwide. In the recent years, numerous studies highlighted the dual role of the gut microbiota in preserving host's health. Gut resident bacteria are able to produce a number of metabolites and bioproducts necessary to protect host's and gut's homeostasis. Conversely, several microbiota subpopulations may expand during pathological dysbiosis and therefore produce high levels of toxins capable, in turn, to trigger both inflammation and tumorigenesis. Importantly, gut microbiota can interact with the host either modulating directly the gut epithelium or the immune system. Numerous gut populating bacteria, called probiotics, have been identified as protective against the genesis of tumors. Given their capability of preserving gut homeostasis, probiotics are currently tested to help to fight dysbiosis in cancer patients subjected to chemotherapy and radiotherapy. Most recently, three independent studies show that specific gut resident species may potentiate the positive outcome of anti-cancer immunotherapy. The highly significant studies, uncovering the tight association between gut microbiota and tumorigenesis, as well as gut microbiota and anti-cancer therapy, are here described. The role of the Lactobacillus rhamnosus GG (LGG), as the most studied probiotic model in cancer, is also reported. Overall, according to the findings here summarized, novel strategies integrating probiotics, such as LGG, with conventional anti-cancer therapies are strongly encouraged.

Type M Resistance to Macrolides Is Due to a Two-Gene Efflux Transport System of the ATP-Binding Cassette (ABC) Superfamily.

The mef(A) gene was originally identified as the resistance determinant responsible for type M resistance to macrolides, a phenotype frequently found in clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes. MefA was defined as a secondary transporter of the major facilitator superfamily driven by proton-motive force. However, when characterizing the mef(A)-carrying elements Tn1207.1 and Φ1207.3, another macrolide resistance gene, msr(D), was found adjacent to mef(A). To define the respective contribution of mef(A) and msr(D) to macrolide resistance, three isogenic deletion mutants were constructed by transformation of a S. pneumoniae strain carrying Φ1207.3: (i) Δmef(A)-Δmsr(D); (ii) Δmef(A)-msr(D); and (iii) mef(A)-Δmsr(D). Susceptibility testing of mutants clearly showed that msr(D) is required for macrolide resistance, while deletion of mef(A) produced only a twofold reduction in the minimal inhibitory concentration (MIC) for erythromycin. The contribution of msr(D) to macrolide resistance was also studied in S. pyogenes, which is the original host of Φ1207.3. Two isogenic strains of S. pyogenes were constructed: (i) FR156, carrying Φ1207.3, and (ii) FR155, carrying Φ1207.3/Δmsr(D). FR155 was susceptible to erythromycin, whereas FR156 was resistant, with an MIC value of 8 µg/ml. Complementation experiments showed that reintroduction of the msr(D) gene could restore macrolide resistance in Δmsr(D) mutants. Radiolabeled erythromycin was retained by strains lacking msr(D), while msr(D)-carrying strains showed erythromycin efflux. Deletion of mef(A) did not affect erythromycin efflux. This data suggest that type M resistance to macrolides in streptococci is due to an efflux transport system of the ATP-binding cassette (ABC) superfamily, in which mef(A) encodes the transmembrane channel, and msr(D) the two ATP-binding domains.

Spread of Vancomycin-Resistant Enterococcus faecium Isolates Despite Validated Infection Control Measures in an Italian Hospital: Antibiotic Resistance and Genotypic Characterization of the Endemic Strain.

An alarming increase of vancomycin-resistant Enterococcus faecium (VREfm) isolates was detected in an Italian referral hospital subjected to policies of infection control validated by the Joint Commission International. Analysis of the population structure of 122 consecutive, nonreplicate VREfm isolates collected over an 18-month period identified a single major clone that spread around the whole hospital, rapidly establishing an endemic state. It belonged to sequence type (ST) 17 and showed a highly multidrug-resistant phenotype, being resistant to all antimicrobial classes for the carriage of several resistance determinants. Furthermore, some strains with decreased susceptibility to daptomycin were detected. Eighteen out of the 122 isolates did not group in the major clone. They showed a low spreading potential inside the hospital wards, even if most of them displayed a multidrug-resistant phenotype and belonged to a hospital-adapted lineage. Causes that led to the VREfm endemic state have not been fully elucidated. However, it is conceivable that the increase in systemic antibiotic consumption and the use of selective digestive tract decontamination, including vancomycin in critically ill patients during the period before 2014, may have played a role in the ST17 clone dissemination, but additional traits conferring high fitness in hospital environment cannot be excluded.

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.

Immunomodulatory Effects of Bifidobacterium longum W11 Produced Exopolysaccharide on Cytokine Production.

BACKGROUND: Health benefits, including immune modulating capability, exerted by Bifidobacterium strains have been attributed to their exopolysaccharides (EPSs). OBJECTIVE: The effects of the purified EPS from B. longum W11 on cytokine production by peripheral blood mononuclear cells (PBMCs) alone or ConA-stimulated were investigated. METHOD: The production of IFN-γ, IL-1ß, IL-6 and IL-10 by PBMCs from healthy adult donors was analysed using purified EPS at two different concentrations (100 µg/mL and 200 µg/mL) and ConA, as an immunopotentiating marker. Moreover, the monosaccharide composition of the EPS from B. longum W11 was detected using HPLC analysis. RESULTS: The results demonstrated the ability of purified EPS to increase the production of the tested cytokines, except IL-10, in ConA-stimulated PBMCs. In not-stimulated-PBMCs, EPS increased the production of IL-6 (at 200 µg/mL) and IL-10 (at 100 µg/mL). The HPLC analysis showed the presence of main monomers, galactose and glucose (ratio 1:1 wt/wt), and small amount of rhamnose. CONCLUSION: The results of this study demonstrate the ability of the EPS produced by B. longum W11 to interact in vitro with the human PBMCs, showing an immune-regulatory profile alone and an immune stimulatory profile in ConA-stimulated PBMCs. This suggests putative applications for the EPS from B. longum W11 in different pathological conditions.

Lactobacillus rhamnosus GG: An Overview to Explore the Rationale of Its Use in Cancer.

Cancer is the second leading cause of death in the western world. In the era of precision medicine, a significant number of cancer patients can be cured with several anti-cancer therapeutic regimens. However, therapy failure may be caused by treatment side effects, such as diarrhea, especially occurring in patients with gastrointestinal or pelvic malignancies. In particular, diarrhea is one of the most frequent gastrointestinal toxicity during cancer treatment and it can result from nearly bot chemo- and radio-therapeutic strategies currently used. Diarrhea has a serious impact on patients' quality of life and treatment dosing and schedule modification due to its severity can negatively influence treatment outcomes. In this context, probiotics may play an interesting role in several human diseases with an inflammatory bowel involvement and, among these, Lactobacillus rhamnosus GG (LGG) is one of the most characterized and utilized. In particular, LGG is able to reverse intestinal dysbiosis and moderate diarrhea. Moreover, preclinical studies have documented its effects in reducing chronic inflammation associated with cancer development. This review summarizes the preclinical results of LGG on cancer cells proliferation and tumor invasion as well as the potential role of LGG use in cancer patients for the prevention and management of diarrhea associated with cancer treatment. Overall, these encouraging data support further investigation on the use of LGG in stratified patients undergoing specific therapeutic protocols, including chemotherapy and pelvic radiotherapy, in order to reduce the development of severe diarrhea and thus improve the adherence to the therapy and patients' quality of life.

Insights and clinical perspectives of daptomycin resistance in Staphylococcus aureus: A review of the available evidence.

The emergence of glycopeptide reduced susceptibility and resistance in Staphylococcus aureus strains is a growing clinical problem that poses significant clinical challenges in treatment. Its development is a complex and novel process involving many subtle physiological changes in the micro-organism. Daptomycin is the first cyclic lipopeptide approved for clinical use. Unlike most other antimicrobials, a trend towards increased daptomycin resistance has not been reported, although several cases of daptomycin non-susceptibility have been reported. The present review will present the available evidence on daptomycin resistance of S. aureus, with particular attention to its development. In addition to a literature overview, we have compiled the reported cases of daptomycin non-susceptibility to shed light on possible clinical mechanisms of resistance. In the 36 reports describing 62 clinical cases, infections caused by meticillin-resistant S. aureus (MRSA) strains with a vancomycin minimum inhibitory concentration (MIC) between 1mg/L and 2mg/L often led to vancomycin treatment failure, which may be associated with the development of non-susceptibility to daptomycin. Additional evidence suggests that underdosage of daptomycin is an important clinical aspect that merits further study. The current analysis highlights the importance of determining the MIC when using vancomycin to treat patients with severe S. aureus infections and that when failure is suspected, testing for heterogeneous vancomycin-intermediate S. aureus (hVISA) may also be necessary. Whilst further investigation is needed, it can be hypothesised that MRSA strains become hVISA during prolonged bacteraemia, which may predispose to the development of daptomycin resistance.

Nucleotide sequence of conjugative prophage Φ1207.3 (formerly Tn1207.3) carrying the mef(A)/msr(D) genes for efflux resistance to macrolides in Streptococcus pyogenes.

Genetic element Φ1207.3 (formerly Tn1207.3) is a prophage of Streptococcus pyogenes which carries the macrolide efflux resistance genes mef(A)/msr(D) and is capable of conjugal transfer among streptococci. Complete nucleotide sequence showed that Φ1207.3 is 52,491 bp in length and contained 58 open reading frames (ORFs). A manual homology-based annotation with functional prediction of the hypothetical gene product was possible only for 34 out of 58 ORFs. Φ1207.3 codes for two different C-methylation systems, several phage structural genes, a lysis cassette (composed by a holin and a peptidoglycan hydrolase), and three site-specific resolvases of the serine recombinase family.

Rapidly fatal hemorrhagic pneumonia and group A Streptococcus serotype M1.

We report 3 cases of fulminant hemorrhagic pneumonia in previously health patients. Sudden-onset hemoptysis and dyspnea developed; all 3 patients and died <12 h later of massive pulmonary bleeding, despite aggressive supportive care. Postmortem analysis showed that the illnesses were caused by group A Streptococcus emm1/sequence type 28 strains.

Genomic diversification of enterococci in hosts: the role of the mobilome.

Enterococci are ubiquitous lactic acid bacteria, possessing a flexible nature that allows them to colonize various environments and hosts but also to be opportunistic pathogens. Many papers have contributed to a better understanding of: (i) the taxonomy of this complex group of microorganisms; (ii) intra-species variability; (iii) the role of different pathogenicity traits; and (iv) some markers related to the character of host-specificity, but the reasons of such incredible success of adaptability is still far from being fully explained. Recently, genomic-based studies have improved our understanding of the genome diversity of the most studied species, i.e., E. faecalis and E. faecium. From these studies, what is becoming evident is the role of the mobilome in adding new abilities to colonize new hosts and environments, and eventually in driving their evolution: specific clones associated with human infections or specific hosts can exist, but probably the consideration of these populations as strictly clonal groups is only partially correct. The variable presence of mobile genetic elements may, indeed, be one of the factors involved in the evolution of one specific group in a specific host and/or environment. Certainly more extensive studies using new high throughput technologies are mandatory to fully understand the evolution of predominant clones and species in different hosts and environments.

Bacteriocin-producing oral streptococci and inhibition of respiratory pathogens.

The use of bacteria as probiotics is in continuous development, thanks to their capacity to maintain or restore a host's natural microbiome by interference with and/or inhibition of other microorganisms mediated by antimicrobial peptide production such as bacteriocins. In the oral cavity, Streptococcus salivarius, a non-pathogenic and predominant oral species, is one of the major bacteriocin producers that is able to coexist in this environment and reduce the frequency of colonization of the main pathogens involved in upper respiratory tract infections. The aim of this study was to screen oral bacteria colonizing healthy children for their use as potential oral probiotics. Eighty-one α-hemolytic streptococci isolated from nasal and/or pharyngeal swabs of 31 healthy children aged between two and twelve years were isolated. Among them, 13 α-hemolytic streptococci were selected for their bacteriocin-like inhibitory activity against potential pathogens. These strains were tested for bacteriocin production and assayed for their capacity to adhere to HEp-2 cell lines. Our data showed that 13 bacteriocin producer strains were able to inhibit different gram-positive pathogens. Among them one strain, S. salivarius 24SMB, deposited as DSM 23307, was selected as a potential oral probiotic, thanks to its safety assessment, ability to inhibit Streptococcus pneumoniae and the absence of virulence and antibiotic resistance genes.