Isolation and Characterization of a Novel Alpha-Hemolytic Streptococcus spp. from the Oral Cavity and Blood of Septicemic Periparturient Immunodeficient Mice.

MISTRG is an immunodeficient mouse strain that expresses multiple human cytokines that support hematopoietic stem cell maintenance and myelopoiesis. While establishing a breeding colony of MISTRG mice in a dedicated barrier room, 6 cases of death or disease occurred in pregnant or postpartum mice. Clinically, this manifested as hunched posture, dyspnea, and 1 case of emaciation with ataxia. Pathologic analysis of 7 mice revealed multisystemic necrosuppurative inflammation variably affecting the uterus and placenta, joints, meninges, inner and middle ears, kidneys, and small intestine. Bacteria cultured from the blood of septic mice were identified with 89% probability by the Vitek 2 identification system as Streptococcus sanguinus with atypical biochemical parameters; the API 20E/NE system fully differentiated the isolates as a novel Streptococcus species. MALDI Biotyper-based mass spectrometry also indicated that the phenotype represented a novel Streptococcus spp. Sequencing revealed that the full-length 16S rRNA gene identity was below 97% with known Streptococcus species, including the 2 closest species Streptococcus acidominimus and Streptococcus azizii. We propose the name Streptococcus murisepticum spp. nov to our novel isolates. All male mice in this colony remained healthy despite their association with diseased female mice. Overall, 19% of the colony carried the novel Streptococcus in their oral cavity, but it could not be detected in feces. The organism was sensitive to amoxicillin, which was administered via drinking water throughout pregnancy and weaning to establish a colony of pathogen-negative future breeders. The colony remained disease-free and culture-negative for Streptococcus murisepticum spp. nov after treatment with amoxicillin. We suspect that oral colonization of MISTRG mice with the novel Streptococcus species and its associated unique pathology in periparturient mice is potentially the principal cause of loss of this strain at several institutions. Therefore, screening the oral cavity for α-hemolytic streptococci followed by targeted antibiotic treatment may be necessary when establishing MISTRG and allied immunodeficient mouse strains.

Oral microbial communities in children, caregivers, and associations with salivary biomeasures and environmental tobacco smoke exposure.

Human oral microbial communities are diverse, with implications for oral and systemic health. Oral microbial communities change over time; thus, it is important to understand how healthy versus dysbiotic oral microbiomes differ, especially within and between families. There is also a need to understand how the oral microbiome composition is changed within an individual including by factors such as environmental tobacco smoke (ETS) exposure, metabolic regulation, inflammation, and antioxidant potential. Using archived saliva samples collected from caregivers and children during a 90-month follow-up assessment in a longitudinal study of child development in the context of rural poverty, we used 16S rRNA gene sequencing to determine the salivary microbiome. A total of 724 saliva samples were available, 448 of which were from caregiver/child dyads, an additional 70 from children and 206 from adults. We compared children's and caregivers' oral microbiomes, performed "stomatotype" analyses, and examined microbial relations with concentrations of salivary markers associated with ETS exposure, metabolic regulation, inflammation, and antioxidant potential (i.e., salivary cotinine, adiponectin, C-reactive protein, and uric acid) assayed from the same biospecimens. Our results indicate that children and caregivers share much of their oral microbiome diversity, but there are distinct differences. Microbiomes from intrafamily individuals are more similar than microbiomes from nonfamily individuals, with child/caregiver dyad explaining 52% of overall microbial variation. Notably, children harbor fewer potential pathogens than caregivers, and participants' microbiomes clustered into two groups, with major differences being driven by Streptococcus spp. Differences in salivary microbiome composition associated with ETS exposure, and taxa associated with salivary analytes representing potential associations between antioxidant potential, metabolic regulation, and the oral microbiome. IMPORTANCE The human oral cavity is a multi-environment habitat that harbors a diversity of microorganisms. This oral microbiome is often transmitted between cohabitating individuals, which may associate oral and systemic health within family members. Furthermore, family social ecology plays a significant role in childhood development, which may be associated with lifelong health outcomes. In this study, we collected saliva from children and their caregivers and used 16S rRNA gene sequencing to characterize their oral microbiomes. We also analyzed salivary biomeasures of environmental tobacco smoke exposure, metabolic regulation, inflammation, and antioxidant potential. We show there are differences in individuals' oral microbiomes mainly due to Streptococcus spp. that family members share much of their microbes, and several bacterial taxa associate with the selected salivary biomeasures. Our results suggest there are large-scale oral microbiome patterns, and there are likely relationships between oral microbiomes and the social ecology of families.

Effect of Lactobacillus delbrueckii subsp. lactis on vaginal radiotherapy for gynecological cancer.

The aim of this study was to evaluate the effect of Lactobacillus delbrueckii subsp. lactis (L.del) on vaginal microbiota (VM) dysbiosis and vaginal radiation injury in gynecologic cancer patients. The inhibitory effects of L.del on cervical cancer cells were also studied in vitro. Gynecologic cancer patients receiving radiotherapy were randomized into control and L.del intervention groups. The control group received radiotherapy, while the intervention group received radiotherapy and L.del intervention (1 capsule/day placed into the deep vagina from the first day of radiotherapy until the end of treatment). Vaginal swab samples were collected on the first day pre-treatment and the last day post-treatment. DNA from 54 patients was extracted and assessed by the 16S rRNA sequencing method. Radiotherapy resulted in vaginal microbiome dysbiosis characterized by increased phylogenetic diversity and increased abundance of Brevundimonas, Streptococcus and Prevotella, but a decreased abundance of Lactobacillus. Level 2 vaginal radiation injury was positively associated with the abundance of Brevundimonas and gram-negative non-fermenting bacteria. Administration of L.del attenuated the reduction of Lactobacillus while also inhibiting the abundance of Streptococcus and Prevotella, thereby ameliorating radiotherapy-related vaginal microbiota dysbiosis. CLD inhibited the in vitro proliferation of SiHa cells by altering the expression of BCL2, HPV16-E6, HPV16-E7, IL6, MAP7, BAX, Caspase-3, Caspase-9 and LTF. In conclusion, L. del application can alleviate radiation-induced vaginal dysbiosis and restore Lactobacillus dominance of the vaginal microbiome. Moreover, CLD was found to inhibit cell growth and promote the apoptosis of SiHa cells in vitro. The registration number for this clinical trial is ChiCTR1900021784.

The ComRS-SigX Pathway Regulates Natural Transformation in Streptococcus ferus.

The ability to take up and incorporate foreign DNA via natural transformation is a well-known characteristic of some species of Streptococcus, and is a mechanism that rapidly allows for the acquisition of antibacterial resistance. Here, we describe that the understudied species Streptococcus ferus is also capable of natural transformation and uses a system analogous to that identified in Streptococcus mutans. S. mutans natural transformation is under the control of the alternative sigma factor sigX (also known as comX), whose expression is induced by two types of peptide signals: CSP (competence stimulating peptide, encoded by comC) and XIP (sigX-inducing peptide, encoded by comS). These systems induce competence via either the two-component signal-transduction system ComDE or the RRNPP transcriptional regulator ComR, respectively. Protein and nucleotide homology searches identified putative orthologs of comRS and sigX in S. ferus, but not homologs of S. mutans blpRH (also known as comDE). We demonstrate that natural transformation in S. ferus is induced by a small, double-tryptophan containing sigX-inducing peptide (XIP), akin to that of S. mutans, and requires the presence of the comR and sigX orthologs for efficient transformation. Additionally, we find that natural transformation is induced in S. ferus by both the native XIP and the XIP variant of S. mutans, implying that cross talk between the two species is possible. This process has been harnessed to construct gene deletions in S. ferus and provides a method to genetically manipulate this understudied species. IMPORTANCE Natural transformation is the process by which bacteria take up DNA and allows for acquisition of new genetic traits, including those involved in antibiotic resistance. This study demonstrates that the understudied species Streptococcus ferus is capable of natural transformation using a peptide-pheromone system like that previously identified in Streptococcus mutans and provides a framework for future studies concerning this organism.

Detection of immunoreactive proteins of Escherichia coli, Streptococcus uberis, and Streptococcus agalactiae isolated from cows with diagnosed mastitis.

Introduction: Mastitis is a widespread mammary gland disease of dairy cows that causes severe economic losses to dairy farms. Mastitis can be caused by bacteria, fungi, and algae. The most common species isolated from infected milk are, among others, Streptococcus spp., and Escherichia coli. The aim of our study was protein detection based on both in silico and in vitro methods, which allowed the identification of immunoreactive proteins representative of the following species: Streptococcus uberis, Streptococcus agalactiae, and Escherichia coli. Methods: The study group included 22 milk samples and 13 serum samples obtained from cows with diagnosed mastitis, whereas the control group constituted 12 milk samples and 12 serum samples isolated from healthy animals. Detection of immunoreactive proteins was done by immunoblotting, while amino acid sequences from investigated proteins were determined by MALDI-TOF. Then, bioinformatic analyses were performed on detected species specific proteins in order to investigate their immunoreactivity. Results: As a result, we identified 13 proteins: 3 (molybdenum cofactor biosynthesis protein B, aldehyde reductase YahK, outer membrane protein A) for E. coli, 4 (elongation factor Tu, tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG, GTPase Obg, glyceraldehyde-3-phosphate dehydrogenase) for S. uberis, and 6 (aspartate carbamoyltransferase, elongation factor Tu, 60 kDa chaperonin, elongation factor G, galactose-6-phosphate isomerase subunit LacA, adenosine deaminase) for S. agalactiae, which demonstrated immunoreactivity to antibodies present in serum from cows with diagnosed mastitis. Discussion: Due to the confirmed immunoreactivity, specificity and localization in the bacterial cell, these proteins can be considered considered potential targets in innovative rapid immunodiagnostic assays for bovine mastitis, however due to the limited number of examined samples, further examination is needed.

Markerless Genome Editing in Competent Streptococci.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.

Abiotrophia spp. and Granulicatella spp. Infective Endocarditis: A Contemporary Perspective.

BACKGROUND: Abiotrophia spp. and Granulicatella spp. are Gram-positive cocci, formerly known as nutritionally variant or deficient Streptococcus. Their role as causative agents of infective endocarditis (IE) is numerically uncertain, as well as diagnostic and clinical management of this infection. The aim of our study is to describe the clinical, microbiological, therapeutic, and prognosis of patients with IE caused by these microorganisms in a large microbiology department. METHODS: Retrospective analysis of all the patients with Abiotrophia spp. and Granulicatella spp. IE registered in our centre in the period 2004-2021. RESULTS: Of the 822 IE in the study period, 10 (1.2%) were caused by Abiotrophia spp. (7) or Granulicatella spp. (3). The species involved were A.defectiva (7), G.adiacens (2) and G.elegans (1). Eight patients were male, their mean age was 46 years and four were younger than 21 years. The most frequent comorbidities were congenital heart disease (4; 40%) and the presence of intracardiac prosthetic material (5; 50%). IE occurred on 5 native valves and 5 prosthetic valve or material. Blood cultures were positive in 8/10 patients, within a mean incubation period of 18.07 hours. In the other two patients, a positive 16SPCR from valve or prosthetic material provided the diagnosis. Surgery for IE was performed in seven patients (70%) and in all cases positive 16S rRNA PCR and sequencing from valve or prosthetic material was demonstrated. Valves and/or prosthetic removed material cultures were positive in four patients. Nine patients received ceftriaxone (4 in monotherapy and 5 in combination with other antibiotics). The mean length of treatment was 6 weeks and IE-associated mortality was 20% at one year follow-up. CONCLUSIONS: Abiotrophia spp. or Granulicatella spp. IE were infrequent but not exceptional in our environment and particularly affected patients with congenital heart disease or prosthetic material. Blood cultures and molecular methods allowed the diagnosis. Most of them required surgery and the associated mortality, in spite of a mean age of 46 years, was high.

Streptococcus ruminicola sp. nov., new species of the Streptococcus bovis/Streptococcus equinus complex (SBSEC) isolated from the rumen of Korean domestic ruminants.

A total of three Gram-positive, and oxidase and catalase-negative facultative anaerobic non-motile bacteria were isolated from the rumen fluid of cows and goats and these strains were designated CNU_G2T, CNU_77-61, and CNU_G3. They grew at 20-45 °C, pH 6.5-7, and 0-6.5% NaCl (w/v). The G + C contents (%) of the three isolates were 37.9, 37.8 and 37.8, respectively. Phylogenomic analysis indicated that these strains were distinct from other Streptococcus species. The average nucleotide identity between the isolates and the closest strain S. infantarius subsp. infantarius ATCC BAA-102T was 94.0-94.5%, while the digital DNA-DNA hybridization (dDDH) values between the isolates and the aforementioned related strain were 58.2-61.4%, respectively. Fatty acid analysis revealed higher proportions of C16:0 (> 28%) in all three isolates, while the proportion of C18:0 was higher in CNU_G2T (25.8%); however, it was less than 12% in all the representing strains used in the study. The C14:0 composition of strains CNU_77-61 (22.1%) and CNU_G3 (24.1%) was higher than that of type strains of CNU_G2T (8.1%). Based on the morphological, biochemical, and molecular phylogenetic features of the three novel isolates, they represent a novel species of the genus Streptococcus, for which we propose as Streptococcus ruminicola sp. nov. The type strain is CNU_G2T (= KCTC 43308T = GDMCC 1.2785T).

Streptococcus humanilactis sp.nov., isolated from healthy nursing mother's breast milk.

Two bacterial strains were isolated from the breast milk of two healthy nursing mothers. The isolates were Gram-positive, catalase-negative, coccus-shaped, chain-forming organisms. Analysis of the 16S rRNA gene sequences of strain IMAU99125T shared 99.7 and 99.6% similarity with Streptococcus mitis ATCC 49456 T and Streptococcus pseudopneumoniae ATCC BAA-960 T, respectively. The nearly complete 16S rRNA gene sequences of IMAU99125T and IMAU99674 strains were very closely related (with only 0.06% difference between them). Sequence analysis of the gyrB and rpoB genes also indicated that IMAU99125T was closely related to S. mitis ATCC 49456 T (94.7% and 97.1%, respectively) and S. pseudopneumoniae ATCC BAA-960 T (94.4% and 97.1%, respectively). Average nucleotide identity (ANI) values between strain IMAU99125T and S. mitis ATCC 49456 T and S. pseudopneumoniae ATCC BAA-960 T, were 93.3% and 92.7%, respectively. Genome-to-genome distance (GGD) values between strain IMAU99125T and S. mitis ATCC 99125 T and S. pseudopneumoniae ATCC BAA-960 T were 53.4% (50.7-56.0) and 50.4% (47.7-53.0), respectively. The major fatty acids of the strain were C16:0 (51.4%). On the basis of the results of phenotypic and phylogenetic analyses, we propose that the two strains be classified as representing a novel species of the genus Streptococcus, namely Streptococcus humanilactis sp.nov. The type strain is IMAU99125T (= GDMCC 1.1876 T = KCTC 21157 T). The genome of Streptococcus humanilactis sp. nov. is comprised of 2,027,143 bp. The DNA G + C content of the strain is 40.0 mol%.

Bacteraemia and infective endocarditis with Streptococcus bovis-Streptococcus equinus-complex: a retrospective cohort study.

BACKGROUND: Streptococcus bovis/equinus complex (SBSEC) comprise several species and subspecies and is a common cause of infective endocarditis (IE). S. gallolyticus subsp. gallolyticus (Sg gallolyticus) accounts for a majority of SBSEC IE, but the risk of IE for other subspecies is largely unknown. We aimed to investigate the clinical presentation of bacteraemia, and proportion of patients with IE in bacteraemia with the most common subspecies. METHODS: A retrospective cohort study of SBSEC-bacteraemia identified in clinical laboratory databases, in Skåne Region, Sweden, 2003-2018. Bacteraemia with Sg gallolyticus, S. gallolyticus subsp. pasteurianus (Sg pasteurianus), S. lutetiensis and S. infantarius subsp. infantarius (Si infantarius) were included. Subspecies was identified by whole genome sequencing. Medical charts were reviewed according to a predetermined protocol, IE was defined by the criteria from European Society of Cardiology. RESULTS: In total, 210 episodes of SBSEC-bacteraemia were included. Definite IE was identified in 28/210 (13%) episodes. Of these, 7/28 (25%) were prosthetic valve-IE, 1/28 (4%) related to a cardiovascular implantable electronic device and 10/28 (36%) required heart valve surgery. The proportions of IE among different subspecies were: Sg gallolyticus 17/52 (33%), Si infantarius 5/31 (16%), Sg pasteurianus 4/83 (5%) and S. lutetiensis 2/44 (5%) (p < 0.001). Sg pasteurianus and S. lutetiensis were more often associated with intra-abdominal- and polymicrobial infection. CONCLUSION: The proportion of IE in SBSEC-bacteraemia varies substantially depending on subspecies. Echocardiography should always be considered in bacteraemia with Sg gallolyticus and Si infantarius, and can sometimes be omitted in bacteraemia with Sg pasteurianus and S. lutetiensis.

Overexpression of angiotensin-converting enzyme 2 contributes to the amelioration of Streptococcus uberis-induced inflammatory injury in mammary epithelial cells.

Streptococcus uberis (S. uberis) is an environmentally important pathogenic bacterium and is the main pathogenic microorganism responsible for mastitis, which causes significant economic losses worldwide. Currently, there is no particularly effective treatment other than antibiotic therapy. Angiotensin-converting enzyme 2 (ACE2) plays an anti-inflammatory as well as an anti-injury role in numerous inflammatory diseases. Therefore, this study aimed to assess the hypothesis that S. uberis-induced mammary epithelial cells injury associated with ACE2, angiotensin II (Ang II) as well as angiotensin 1-7 (Ang-(1-7)) imbalance and that overexpression of ACE2 can repair S. uberis-induced mammary epithelial cells injury. We observed that the expression level of ACE2 was significantly downregulated after treatment of EpH4-Ev cells with S. uberis. Next, this assay verified the role of ACE2 in S. uberis-induced inflammatory injury in EpH4-Ev cells by overexpressing the ACE2 gene as well as its silencing. The results showed that overexpression of the ACE2 gene significantly activated the interleukin-10/signal transducer and activator of transcription 3/suppressors-of-cytokine-signaling 3 (IL-10/STAT3/SOCS3) pathway, thereby inhibiting the nuclear factor-κB (NF-κB) as well as pyroptosis pathways. Furthermore, overexpression of the ACE2 gene reversed the downregulation of zonula occludens 1 (ZO-1), Occludin, Claudin-1, and Claudin-2 caused by S. uberis, suggesting that ACE2 could promote to repair the blood-milk barrier. However, siRNA silencing of the ACE2 gene produced the opposite effect. These results suggest that ACE2 ameliorates S. uberis-induced mammary epithelial cells injury. AVAILABILITY OF DATA: All data generated or analyzed during this study are included within the article and its additional information file.

Fecal Streptococcus Alteration Is Associated with Gastric Cancer Occurrence and Liver Metastasis.

The gut microbiome plays an indispensable role in the occurrence and progression of various diseases. However, its ability to predict gastric cancer (GC) and liver metastasis (GCLM) has not been fully identified. Fecal samples were collected from 49 GC patients (cancer group [group C]) and 49 healthy people (normal group [group N]) between 4 July 2020 and 9 March 2021. Furthermore, 26 patients with metastatic GC were divided into a liver metastatic group (group L) (n = 13) and a non-liver-metastatic group (group M) (n = 13). DNA was extracted, and 16S rRNA gene sequencing was performed. SPSS was used for statistical analyses, and all bioinformatics analyses were based on QIIME2. P values of <0.05 were considered statistically significant. The microbial richness and diversity in group C were higher than those in group N, and there were significant differences in species compositions between the two groups. Streptococcus, enriched in groups C and L by linear discriminant analysis (LDA) effect size (LEfSe) and further identified by a random forest (RF) model, enhances its potential as a biomarker for GC and GCLM. Functional gene and metabolic pathway analyses showed that d-galacturonate degradation pathway II was of great importance in the occurrence and development of GC. Streptococcus has the potential ability to predict GC and GCLM, which is critical for the early diagnosis of GC and GCLM. IMPORTANCE The gut microbiome plays an indispensable role in the occurrence and progression of various diseases. However, its ability to predict gastric cancer (GC) and liver metastasis (GCLM) has not been fully identified. We retrospectively analyzed 49 untreated GC patients and 49 matched healthy people between 4 July 2020 and 9 March 2021. By extracting DNA from their fecal samples and sequencing the 16S rRNA gene, we found that Streptococcus alteration was strongly associated with GC occurrence and liver metastasis, which might be a potential biomarker in predicting GC and GCLM, and the results of this study are helpful in providing ideas for the early diagnosis and treatment of GC.

RNase Z Oxidative Degradation Impedes tRNA Maturation and is Involved in Streptococcal Translation Regulation in Response to Oxidative Stress.

When encountering oxidative stress, organisms selectively upregulate antioxidant genes and simultaneously suppress the translation of most other proteins. Eukaryotes employ multiple strategies to adjust translation at both the initiation and elongation stages; however, how prokaryotes modulate translation under oxidative stress remains unclear. Here, we report that upon hydrogen peroxide (H2O2) challenge, Streptococcus oligofermentans reduced translation via RNase Z (So-RNaseZ) oxidative degradation, thus hindering tRNA maturation. S. oligofermentans encodes all CCA-less tRNAs that require So-RNaseZ for 3' end maturation. A combination of nonreducing SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC/MS-MS) assays demonstrated that H2O2 oxidation induced Cys38-Cys149 disulfide linkages in recombinant So-RNaseZ protein, and serine substitution of Cys38 or Cys149 abolished these disulfide linkages. Consistently, redox Western blotting also determined intramolecular disulfide-linked So-RNaseZ in H2O2-treated S. oligofermentans cells. The disulfide-linked So-RNaseZ and monomer were both subject to proteolysis, whereas C149S mutation alleviated oxidative degradation of So-RNaseZ, suggesting that H2O2-mediated disulfide linkages substantially contributed to So-RNaseZ degradation. Accordingly, Northern blotting determined that tRNA precursor accumulation and mature tRNA species decrease in H2O2-treated S. oligofermentans. Moreover, reduced overall protein synthesis, as indicated by puromycin incorporation, and retarded growth of S. oligofermentans occurred in an H2O2 concentration-dependent manner. Overexpression of So-RNaseZ not only elevated tRNA precursor processing and protein synthesis but also partly rescued H2O2-suppressed S. oligofermentans growth. Moreover, So-RNaseZ oxidative degradation-mediated translation repression elevated S. oligofermentans survival under high H2O2 stress. Therefore, this work found that So-RNaseZ oxidative degradation-impeded tRNA maturation contributes to streptococcal translation repression and provides the oxidative stress adaptability for S. oligofermentans. IMPORTANCE Translation regulation is a common strategy used by organisms to reduce oxidative damage. Catalase-negative streptococci produce as well as tolerate high levels of H2O2. This work reports a novel translation regulation mechanism employed by Streptococcus oligofermentans in response to H2O2 challenge, in which the key tRNA endonuclease So-RNaseZ is oxidized to form Cys38-Cys149 disulfide linkages and both the disulfide-linked So-RNaseZ and monomers are subject to proteolysis; thus, tRNA maturation, protein translation, and growth are all suppressed. Notably, So-RNaseZ oxidative degradation-mediated translation repression offers oxidative adaptability to S. oligofermentans and enhances its survival against high H2O2 challenge. So-RNaseZ orthologs and H2O2-sensitive cysteines (Cys38 and Cys149) are widely distributed in Streptococcus and Lactococcus species genomes, which also encode all CCA-less tRNAs and lack catalase. Therefore, RNase Z oxidative degradation-based translation regulation could be widely employed by these lactic acid bacteria, including pathogenic streptococci, to cope with H2O2.

Pathogenomics of Streptococcus ilei sp. nov., a newly identified pathogen ubiquitous in human microbiome.

Viridans group streptococci are a serious health concern because most of these bacteria cause life-threatening infections, especially in immunocompromised and hospitalized individuals. We focused on two alpha-hemolytic Streptococcus strains (I-G2 and I-P16) newly isolated from an ileostomy effluent of a colorectal cancer patient. We examined their pathogenic potential by investigating their prevalence in human and assessing their pathogenicity in a mouse model. We also predicted their virulence factors and pathogenic features by using comparative genomic analysis and in vitro tests. Using polyphasic and systematic approaches, we identified the isolates as belonging to a novel Streptococcus species and designated it as Streptococcus ilei. Metagenomic survey based on taxonomic assignment of datasets from the Human Microbiome Project revealed that S. ilei is present in most human population and at various body sites but is especially abundant in the oral cavity. Intraperitoneal injection of S. ilei was lethal to otherwise healthy C57BL/6J mice. Pathogenomics and in vitro assays revealed that S. ilei possesses a unique set of virulence factors. In agreement with the in vivo and in vitro data, which indicated that S. ilei strain I-G2 is more pathogenic than strain I-P16, only the former displayed the streptococcal group A antigen. We here newly identified S. ilei sp. nov., and described its prevalence in human, virulence factors, and pathogenicity. This will help to prevent S. ilei strain misidentification in the future, and improve the understanding and management of streptococcal infections.

Transcriptome analysis unveils survival strategies of Streptococcus parauberis against fish serum.

Streptococcus parauberis is an important bacterial fish pathogen that causes streptococcosis in a variety of fish species including the olive flounder. Despite its importance in the aquaculture industry, little is known about the survival strategy of S. parauberis in the host. Therefore, the objective of this study was to produce genome-wide transcriptome data and identify key factors for the survival of S. parauberis SPOF3K in its host. To this end, S. parauberis SPOF3K was incubated in olive flounder serum and nutrient-enriched media as a control. Although S. parauberis SPOF3K proliferated in both culture conditions, the transcriptomic patterns of the two groups were very different. Interestingly, the expression levels of genes responsible for the replication of an S. parauberis plasmid in the presence of olive flounder serum were higher than those in the absence of olive flounder serum, indicating that this plasmid may play an important role in the survival and proliferation of S. parauberis in the host. Several ATP-binding cassette transporters known to transport organic substrates (e.g., biotin and osmoprotectants) that are vital for bacterial survival in the host were significantly up-regulated in S. parauberis cultured in serum. In addition, groEL, dnaK operon, and members of the clp protease family, which are known to play important roles in response to various stressors, were up-regulated in S. parauberis incubated in serum, thus limiting damage and facilitating cellular recovery. Moreover, important virulence factors including the hyaluronic acid capsule (has operon), sortase A (srtA), C5a peptidase (scp), and peptidoglycan O-acetyltransferase (oatA) were significantly upregulated in S. paraubers in serum. These results indicate that S. paraubers can resist and evade the humoral immune responses of fish. The transcriptomic data obtained in this study provide a better understanding of the mode of action of S. parauberis in fish.

Trapping a cross-linked lysine-tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB.

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon-carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys-Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]2+ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys-Trp radical (Lys-Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys-Trp• intermediate by the redox-tuned [4Fe-4S]2+ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon-carbon bond-forming reactions.

Quorum Sensing in Streptococcus mutans Regulates Production of Tryglysin, a Novel RaS-RiPP Antimicrobial Compound.

The genus Streptococcus encompasses a large bacterial taxon that commonly colonizes mucosal surfaces of vertebrates and is capable of disease etiologies originating from diverse body sites, including the respiratory, digestive, and reproductive tracts. Identifying new modes of treating infections is of increasing importance, as antibiotic resistance has escalated. Streptococcus mutans is an important opportunistic pathogen that is an agent of dental caries and is capable of systemic diseases such as endocarditis. As such, understanding how it regulates virulence and competes in the oral niche is a priority in developing strategies to defend from these pathogens. We determined that S. mutans UA159 possesses a bona fide short hydrophobic peptide (SHP)/Rgg quorum-sensing system that regulates a specialized biosynthetic operon featuring a radical-SAM (S-adenosyl-l-methionine) (RaS) enzyme and produces a ribosomally synthesized and posttranslationally modified peptide (RiPP). The pairing of SHP/Rgg regulatory systems with RaS biosynthetic operons is conserved across streptococci, and a locus similar to that in S. mutans is found in Streptococcus ferus, an oral streptococcus isolated from wild rats. We identified the RaS-RiPP product from this operon and solved its structure using a combination of analytical methods; we term these RiPPs tryglysin A and B for the unusual Trp-Gly-Lys linkage. We report that tryglysins specifically inhibit the growth of other streptococci, but not other Gram-positive bacteria such as Enterococcus faecalis or Lactococcus lactis We predict that tryglysin is produced by S. mutans in its oral niche, thus inhibiting the growth of competing species, including several medically relevant streptococci.IMPORTANCE Bacteria interact and compete with a large community of organisms in their natural environment. Streptococcus mutans is one such organism, and it is an important member of the oral microbiota. We found that S. mutans uses a quorum-sensing system to regulate production of a novel posttranslationally modified peptide capable of inhibiting growth of several streptococcal species. We find inhibitory properties of a similar peptide produced by S. ferus and predict that these peptides play a role in interspecies competition in the oral niche.

Immunomodulatory streptococci that inhibit CXCL8 secretion and NFκB activation are common members of the oral microbiota.

Introduction. Oral tissues are generally homeostatic despite exposure to many potential inflammatory agents including the resident microbiota. This requires the balancing of inflammation by regulatory mechanisms and/or anti-inflammatory commensal bacteria. Thus, the levels of anti-inflammatory commensal bacteria in resident populations may be critical in maintaining this homeostatic balance.Hypothesis/Gap Statement. The incidence of immunosuppressive streptococci in the oral cavity is not well established. Determining the proportion of these organisms and the mechanisms involved may help to understand host-microbe homeostasis and inform development of probiotics or prebiotics in the maintenance of oral health.Aim. To determine the incidence and potential modes of action of immunosuppressive capacity in resident oral streptococci.Methodology. Supragingival plaque was collected from five healthy participants and supragingival and subgingival plaque from five with gingivitis. Twenty streptococci from each sample were co-cultured with epithelial cells±flagellin or LL-37. CXCL8 secretion was detected by ELISA, induction of cytotoxicity in human epithelial cells by lactate dehydrogenase release and NFκB-activation using a reporter cell line. Bacterial identification was achieved through partial 16S rRNA gene sequencing and next-generation sequencing.Results. CXCL8 secretion was inhibited by 94/300 isolates. Immunosuppressive isolates were detected in supragingival plaque from healthy (4/5) and gingivitis (4/5) samples, and in 2/5 subgingival (gingivitis) plaque samples. Most were Streptococcus mitis/oralis. Seventeen representative immunosuppressive isolates all inhibited NFκB activation. The immunosuppressive mechanism was strain specific, often mediated by ultra-violet light-labile factors, whilst bacterial viability was essential in certain species.Conclusion. Many streptococci isolated from plaque suppressed epithelial cell CXCL8 secretion, via inhibition of NFκB. This phenomenon may play an important role in oral host-microbe homeostasis.

Intracellular Invasion Ability and Associated Microbiological Characteristics of Streptococcus canis in Isolates from Japan.

This study evaluated the cell invasion ability (CIA) of Streptococcus canis isolates, and clarified the relationship between high-frequency CIA and its microbiological features. Of the companion animal-origin isolates (n = 117) that were obtained in 2017, 40 isolates were randomly selected with the host information, with two human blood-origin isolates included. CIA was measured using human colon carcinoma epithelium and the hemolytic activity (HA) using sheep blood, along with S. canis M-like protein (SCM) allele typing, sequence type (ST) determination, and antimicrobial resistance (AMR) phenotyping/genotyping. CIA measurements revealed that 19 and 24 isolates had high- and low-frequencies, respectively. HA assessment revealed that 24 and 19 isolates were categorized as high- and low- level, respectively. No difference was observed in the high-/low-level HA between the high- /low-frequency CIA populations. A significant difference was found in the high-/low-frequency CIA between the SCM group I/II populations. Additionally, a significantly higher CIA was found in the SCM allele type 10/type 11 than in the others. A significant association was observed between high-frequency CIA and the ST21/ST41 populations. No difference was found in the high-/low-frequency CIA between the presence and absence of the AMR phenotype/genotype. These observations suggest a relationship between high-frequency CIA and its microbiological characteristics (SCM allele type 10/type 11 or ST21/ST41).

Proteins produced by Streptococcus species in the lower respiratory tract can modify antiviral responses against influenza virus in respiratory epithelial cells.

Seasonal influenza spreads during winter in temperate countries. Primary viral pneumoniae resulting from aggravation triggers acute respiratory distress syndrome, which is a serious respiratory disorder. We have identified a unique pattern of lung microbiota in patients with the syndrome. In this study, we hypothesized that the unique microbiota was also associated with primary influenza viral pneumoniae. Bacterial culture supernatants of Streptococcus oralis and Streptococcus mitis detected from the patients significantly increased viral replication (maximum 10-fold increase) in lung epithelial cells. Our results suggest that the lung environment microbiota is significantly involved in viral replication.