Molecular characterization of a novel putative pathogen, Streptococcus nakanoensis sp. nov., isolated from sputum culture.

Reports of novel species of α-hemolytic Streptococcus have increased recently. However, limited information exists regarding the pathogenicity of these species, with the exception of Streptococcus pneumoniae and Streptococcus pseudopneumoniae. In this study, a quinolone-resistant α-Streptococcus strain, MTG105, was isolated from the sputum of a patient with pneumonia. This strain was first identified as S. pneumoniae at the hospital laboratory; however, it exhibited unique genetic features upon further analysis. Digital DNA-DNA hybridization and average nucleotide identity based on BLAST values from whole-genome sequencing revealed MTG105 to be a novel species closely related to S. pseudopneumoniae. Although MTG105 carried two copies of the pneumolysin gene, similar to S. pseudopneumoniae, this isolate exhibited susceptibility to optochin under both aerobic and 5% CO2 conditions. Notably, no biochemical features could be used to definitively identify this species. In an infection assay using organotypic lung tissue models, MTG105 induced epithelial damage comparable to that of S. pneumoniae and S. pseudopneumoniae, possibly suggesting its potential as a pathogenic α-Streptococcus. The natural transformation abilities of Streptococcus species facilitate their exchange of genes within the same genus, resulting in the existence of species with increasingly more diverse genome structures. Therefore, the identification of this species highlights the importance of monitoring the emergence of novel species exhibiting virulence and/or multidrug resistance. This isolate was proposed as a novel species, designated Streptococcus nakanoensis sp. nov. The type strain was MTG 105T (= JCM 35953T = CCUG 76894T). IMPORTANCE: The genus Streptococcus encompasses a wide range of bacteria with more than 60 species. Recently, there has been a notable increase in reports of novel species of α-Streptococcus based on genomic analysis data. However, limited information exists regarding the pathogenicity of these species. In this study, a quinolone-resistant α-hemolytic Streptococcus strain, MTG105, was isolated from a patient with pneumonia. Genetic analysis revealed that this species was a novel species closely related to S. pseudopneumoniae. In an infection assay using organotypic lung tissue models, MTG105 induced epithelial damage comparable to that caused by S. pneumoniae and S. pseudopneumoniae, strongly suggesting its potential as a pathogenic α-Streptococcus. The natural transformation abilities of Streptococcus species facilitate gene exchange within the same genus, leading to the emergence of species with increasingly diverse genome structures. Therefore, the identification of this species underscores the importance of monitoring the emergence of novel species exhibiting virulence and/or multidrug resistance.

Lapidilactobacillus salsurivasis sp. nov., Secundilactobacillus muriivasis sp. nov., and Streptococcus parasalivarius sp. nov., isolated from Traditional Chinese Pickle.

Four Gram-stain-positive bacterial strains (designated 475-2T, 46-6BT, 778-2T and A810-3), isolated from traditional Chinese pickle, were characterized using a polyphasic taxonomic approach. Strain 475-2T was most closely related to the type strain of Lapidilactobacillus achengensis, having 99.9% 16S rRNA gene sequence similarity, 94.1-95.1% average nucleotide identity (ANI) and 57.6% digital DNA-DNA hybridization (dDDH) values. Strain 46-6BT was most closely related to the type strain of Secundilactobacillus similis, having 99.8% 16S rRNA gene sequence similarity, 94.3-94.9% ANI and 58.9-59.2% dDDH values. Strains 778-2T and A810-3 were phylogenetically related to the type strains of Streptococcus salivarius, Streptococcus thermophilus and Streptococcus vestibularis, having 99.7-99.9% 16S rRNA gene sequence similarities, 89.1-94.4% ANI and 39.0-55.5% dDDH values. Based upon the data obtained in the present study, three novel species, Lapidilactobacillus salsurivasis sp. nov., Secundilactobacillus muriivasis sp. nov. and Streptococcus parasalivarius sp. nov., are proposed and the type strains are 475-2T (= JCM 36613T = CCTCC AB 2023258T = LMG 33412T), 46-6BT (= JCM 36612T = CCTCC AB 2023259T = LMG 33411T) and 778-2T (= JCM 36614T = CCTCC AB 2023257T = LMG 33413T), respectively.

Extraction of high-molecular-weight DNA from Streptococcus spp. for nanopore sequencing in resource-limited settings.

The long-read sequencing platform MinION, developed by Oxford Nanopore Technologies, enables the sequencing of bacterial genomes in resource-limited settings, such as field conditions or low- and middle-income countries. For this purpose, protocols for extracting high-molecular-weight DNA using nonhazardous, inexpensive reagents and equipment are needed, and some methods have been developed for gram-negative bacteria. However, we found that without modification, these protocols are unsuitable for gram-positive Streptococcus spp., a major threat to fish farming and food security in low- and middle-income countries. Multiple approaches were evaluated, and the most effective was an extraction method using lysozyme, sodium dodecyl sulfate, and proteinase K for lysis of bacterial cells and magnetic beads for DNA recovery. We optimized the method to consistently achieve sufficient yields of pure high-molecular-weight DNA with minimal reagents and time and developed a version of the protocol which can be performed without a centrifuge or electrical power. The suitability of the method was verified by MinION sequencing and assembly of 12 genomes of epidemiologically diverse fish-pathogenic Streptococcus iniae and Streptococcus agalactiae isolates. The combination of effective high-molecular-weight DNA extraction and MinION sequencing enabled the discovery of a naturally occurring 15 kb low-copy number mobilizable plasmid in S. iniae, which we name pSI1. We expect that our resource-limited settings-adapted protocol for high-molecular-weight DNA extraction could be implemented successfully for similarly recalcitrant-to-lysis gram-positive bacteria, and it represents a method of choice for MinION-based disease diagnostics in low- and middle-income countries.

Streptococcus raffinosi sp. nov., isolated from human breast milk samples.

Novel Gram-positive, catalase-negative, α-haemolytic cocci were isolated from breast milk samples of healthy mothers living in Hanoi, Vietnam. The 16S rRNA gene sequences of these strains varied by 0-2 nucleotide polymorphisms. The 16S rRNA gene sequence of one strain, designated as BME SL 6.1T, showed the highest similarity to those of Streptococcus salivarius NCTC 8618T (99.4 %), Streptococcus vestibularis ATCC 49124T (99.4 %), and Streptococcus thermophilus ATCC 19258T (99.3 %) in the salivarius group. Whole genome sequencing was performed on three selected strains. Phylogeny based on 631 core genes clustered the three strains into the salivarius group, and the strains were clearly distinct from the other species in this group. The average nucleotide identity (ANI) value of strain BME SL 6.1T exhibited the highest identity with S. salivarius NCTC 8618T (88.4 %), followed by S. vestibularis ATCC 49124T (88.3 %) and S. thermophilus ATCC 19258T (87.4 %). The ANI and digital DNA-DNA hybridization values between strain BME SL 6.1T and other species were below the cut-off value (95 and 70 %, respectively), indicating that it represents a novel species of the genus Streptococcus. The strains were able to produce α-galactosidase and acid from raffinose and melibiose. Therefore, we propose to assign the strains to a new species of the genus Streptococcus as Streptococcus raffinosi sp. nov. The type strain is BME SL 6.1T (=VTCC 12812T=NBRC 116368T).

Streptococcus taoyuanensis sp. nov., a Novel Species Isolated from a Patient with Bacteremia.

Streptococcus spp. are important opportunistic pathogen of bacteremia in both immunocompetent and immunosuppressed patients. A streptococcal strain, designated ST2T, was isolated from the blood specimen of a bacteremic patient. Comparative analyses of 16S rRNA, rpoB and groEL gene sequences demonstrated that the novel strain ST2T is a member of the genus Streptococcus. Based on of 16S rRNA gene sequence similarities, the type strains of Streptococcus (S.) parasanguinis (99.2%), S. ilei (98.8%), S. oralis subsp. oralis (97.6%), S. australis (97.5%) and S. sanguinis (97.5%) were the closest neighbours to strain ST2T. The housekeeping gene sequences (rpoB and groEL) similarities of strain ST2T to these closely related type strains were 80.4-97.4%, respectively. The complete draft genome of strain ST2T consisted of 2,155,906 bp with a G + C content of 42.0%. Strain ST2T has an average nucleotide identity (ANI) value of 94.1 and 81.3% with S. parasanguinis ATCC 15912T and S. ilei I-G2T, respectively. The highest in silico DNA-DNA hybridization value with respect to the closest species S. parasanguinis was 55.6%, below the species cut-off of 70% hybridization. The primary cellular fatty acids of strain ST2T were C16:0, C18:1 ω9c, C18:0 and C14:0. Based on biochemical criteria and molecular genetic evidence, it is proposed that strain ST2T be assigned to a new species of the genus Streptococcus as Streptococcus taoyuanensis sp. nov. The type strain of Streptococcus taoyuanensis is ST2T (=NBRC 115928T = BCRC 81374T) as the type strain.

Streptococcus dysgalactiae subsp. equisimilis infection and its intersection with Streptococcus pyogenes.

SUMMARYStreptococcus dysgalactiae subsp. equisimilis (SDSE) is an increasingly recognized cause of disease in humans. Disease manifestations range from non-invasive superficial skin and soft tissue infections to life-threatening streptococcal toxic shock syndrome and necrotizing fasciitis. Invasive disease is usually associated with co-morbidities, immunosuppression, and advancing age. The crude incidence of invasive disease approaches that of the closely related pathogen, Streptococcus pyogenes. Genomic epidemiology using whole-genome sequencing has revealed important insights into global SDSE population dynamics including emerging lineages and spread of anti-microbial resistance. It has also complemented observations of overlapping pathobiology between SDSE and S. pyogenes, including shared virulence factors and mobile gene content, potentially underlying shared pathogen phenotypes. This review provides an overview of the clinical and genomic epidemiology, disease manifestations, treatment, and virulence determinants of human infections with SDSE with a particular focus on its overlap with S. pyogenes. In doing so, we highlight the importance of understanding the overlap of SDSE and S. pyogenes to inform surveillance and disease control strategies.

A rare case report of meningoencephalitis caused by Streptococcus porcinus.

Acute pyogenic meningitis is a medical emergency. Bacteria are the major causative agents of pyogenic meningitis with Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis being the most common. Here, we describe a case of bacterial meningoencephalitis caused by Streptococcus porcinus. To our knowledge this is the first case described in literature. The patient was treated with ceftriaxone and supportive treatment.

Identification and characterization of a novel α-haemolytic streptococci, Streptococcus parapneumoniae sp. nov., which caused bacteremia with pyelonephritis.

OBJECTIVES: We report a case of bacteremia with pyelonephritis in an adult male with an underlying disease caused by α-hemolytic streptococci. α-Hemolytic streptococci were isolated from blood, but it was challenging to identify its species. This study aimed to characterize the causative bacterium SP4011 and to elucidate its species. METHODS: The whole-genome sequence and biochemical characteristics of SP4011 were determined. Based on the genome sequence, phylogenetic analysis was performed with standard strains of each species of α-hemolytic streptococci. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were calculated. RESULTS: SP4011 showed optochin susceptibility and bile solubility, but did not react with pneumococcal omni antiserum. Phylogenetic analysis of the whole-genome sequence showed that SP4011 clustered with S. pneumoniae and S. pseodopneumoniae and was most closely related to S. pseodopneumoniae. Genomic analysis revealed that ANI and dDDH values between SP4011 and S. pseodopneumoniae were 94.0 % and 56.0 %, respectively, and between SP4011 and S. pneumoniae were 93.3 % and 52.2 %, respectively. Biochemical characteristics also showed differences between SP4011 and S. pseodopneumoniae and between SP4011 and S. pneumoniae. These results indicate that SP4011 is a novel species. CONCLUSION: Our findings indicate that SP4011 is a novel species of the genus Streptococcus. SP4011 has biochemical characteristics similar to S. pneumoniae, making it challenging to differentiate and requiring careful clinical diagnosis. This isolate was proposed to be a novel species, Streptococcus parapneumoniae sp. nov. The strain type is SP4011T (= JCM 36068T = KCTC 21228T).

Molecular and phenotypic identification of bacterial species isolated from cows with mastitis from three regions of Poland.

BACKGROUND: Bovine mastitis is a widespread disease affecting dairy cattle worldwide and it generates substantial losses for dairy farmers. Mastitis may be caused by bacteria, fungi or algae. The most common species isolated from infected milk are, among others, Streptococcus spp., Escherichia coli, Staphylococcus aureus and non-aureus staphylococci and mammaliicocci. The aim of this paper is to determine the frequency of occurrence of bacterial species in milk samples from cows with mastitis from three regions of Poland: the north-east, the south-west and the south. To this end 203 milk samples taken from cows with a clinical form (CM) of mastitis (n = 100) and healthy animals (n = 103) were examined, which included culture on an appropriate medium followed by molecular detection of E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis, as one of the most common species isolated from mastitis milk. RESULTS: The results obtained indicated that S. uberis was the most commonly cultivated CM species (38%, n = 38), followed by S. aureus (22%, n = 22), E. coli (21%, n = 21) and S. agalactiae (18%, n = 18). Similar frequencies in molecular methods were obtained for S. uberis (35.1%) and S. aureus (28.0%). The variation of sensitivity of both methods may be responsible for the differences in the E. coli (41.0%, p = 0.002) and S. agalactiae (5.0%, p = 0.004) detection rates. Significant differences in composition of species between three regions of Poland were noted for E. coli incidence (p < 0.001), in both the culture and molecular methods, but data obtained by the PCR method indicated that this species was the least common in north-eastern Poland, while the culture method showed that in north-eastern Poland E. coli was the most common species. Significant differences for the molecular method were also observed for S. uberis (p < 0.001) and S. aureus (p < 0.001). Both species were most common in southern and south-western Poland. CONCLUSIONS: The results obtained confirm the need to introduce rapid molecular tests for veterinary diagnostics, as well as providing important epidemiological data, to the best of our knowledge data on Polish cows in selected areas of Poland is lacking.

Streptococcus hohhotensis sp. nov., isolated from the breast milk of a healthy woman.

Human breast milk contains lactic acid bacteria (LAB), which have an important influence on the composition of the intestinal microbia of infants. In this study, one strain of an α-hemolytic species of the genus Streptococcus, IMAU99199T, isolated from the breast milk of a healthy nursing mother in Hohhot city PR China, was studied to characterise its taxonomic status using phenotypic and molecular taxonomic methods. The results indicated that it represented a member of the mitis-suis clade, pneumoniae subclade of the genus Streptococcus. It is a Gram-stain-positive, catalase-negative and oxidase-negative bacterium, and the cells are globular, paired or arranged in short chains. The results of a phylogenetic analysis of its 16S rRNA gene and two housekeeping genes (gyrB and rpoB) placed it in the genus Streptococcus. A phylogenetic tree based on 135 single-copy genes sequences indicated that IMAU99199T formed a closely related branch well separated from 'Streptococcus humanilactis' IMAU99125, 'Streptococcus bouchesdurhonensis' Marseille Q6994, Streptococcus mitis NCTC 12261T, 'Streptococcus vulneris' DM3B3, Streptococcus toyakuensis TP1632T, Streptococcus pseudopneumoniae ATCC BAA-960T and Streptococcus pneumoniae NCTC 7465T. IMAU99199T and 'S. humanilactis' IMAU99125 had the highest average nucleotide identity (93.7 %) and digital DNA-DNA hybridisation (55.3 %) values, which were below the accepted thresholds for novel species. The DNA G+C content of the draft genome of IMAU99199T was 39.8 %. The main cellular fatty acids components of IMAU99199T were C16 : 0 and C16 : 1ω7. It grew at a temperature range of 25-45 °C (the optimum growth temperature was 37 °C) and a pH range of 5.0-8.0 (the optimum growth pH was 7.0). These data indicate that strain IMAU99199T represents a novel species in the genus Streptococcus, for which the name Streptococcus hohhotensis sp. nov. is proposed. The type strain is IMAU99199T (=GDMCC 1.1874T=KCTC 21155T).

Streptococcus equi subsp. zooepidemicus: High molecular diversity of Argentinian strains isolated from mares with endometritis.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a mucosal commensal of the lower genital tract in horses and is the most isolated bacterium causing endometritis in mares. The aim of this study was to determine the molecular diversity of S. zooepidemicus obtained from endometritis in mares in Buenos Aires province, Argentina. Thirty isolates obtained from the uterus of mares in 2005 and 2017 were studied. The MLST scheme was applied to identify the Argentinian genotypes and the clonal relationships and patterns of evolutionary descent were identified using the eBURST algorithm - goeBURST. Twenty six different Sequence types (STs) were identified, being only 11 of them previously reported in horses and also, from several host species and tissues. The other 15 STs were reported in Argentinian reproductive strains of mares in our study for the first time. The genotypes obtained from uterus in Argentina were not evenly distributed when all the published S. zooepidemicus STs were analysed, thus, it was not possible to establish that the same lineage circulates in our equine population. The fact that the identified genotypes were also reported in other countries, diverse samples and host species suggest that there is not a host, and an anatomical niche adaptation. Finally, the isolation of the same genotype in the vagina/clitoris and the uterus of the same mare highlights the versatility of S. zooepidemicus and its role as an opportunistic pathogen.

ß-Lactam Susceptibility of Streptococcus dysgalactiae subsp. equisimilis.

All clinical isolates of Streptococcus dysgalactiae subsp. equisimilis (SDSE) are considered susceptible to ß-lactams, the first-line drugs used to treat SDSE infections. However, given that penicillin-non-susceptible SDSE strains have been isolated in Denmark, in this study, we aimed to identify ß-lactam-non-susceptible clinical isolates of SDSE in Japan. In 2018, we collected 150 clinical isolates of S. dysgalactiae, and species identification was performed using a Rapid ID Strep API kit. The minimum inhibitory concentrations (MIC) of six ß-lactams (penicillin G, oxacillin, ceftizoxime, ceftibuten, cefoxitin, and cefaclor) were determined for the 85 clinical isolates identified as SDSE using the agar dilution method standardized by the Clinical & Laboratory Standards Institute. The MIC ranges of penicillin G, oxacillin, ceftizoxime, ceftibuten, cefoxitin, and cefaclor were 0.007-0.06, 0.03-0.12, 0.015-0.06, 0.25-2, 0.12-2, and 0.06-0.5 µg/mL, respectively. None of the clinical isolates of SDSE were non-susceptible to penicillin G, indicating that all 85 clinical isolates of SDSE were susceptible to ß-lactams. Our findings indicate that almost all clinical isolates of SDSE, from several prefectures of Japan, are still susceptible to ß-lactams. Nevertheless, there remains a need for continuous and careful monitoring of drug susceptibility among clinical SDSE isolates in Japan.

Optimization and Evaluation of the 30S-S11 rRNA Gene for Taxonomic Profiling of Oral Streptococci.

Dental caries is a multifactorial disease driven by interactions between the highly complex microbial biofilm community and host factors like diet, oral hygiene habits, and age. The oral streptococci are one of the most dominant members of the plaque biofilm and are implicated in disease but also in maintaining oral health. Current methods used for studying the supragingival plaque community commonly sequence portions of the16S rRNA gene, which often cannot taxonomically resolve members of the streptococcal community past the genus level due to their sequence similarity. The goal of this study was to design and evaluate a more reliable and cost-effective method to identify oral streptococci at the species level by applying a new locus, the 30S-S11 rRNA gene, for high-throughput amplicon sequencing. The study results demonstrate that the newly developed single-copy 30S-S11 gene locus resolved multiple amplicon sequence variants (ASVs) within numerous species, providing much improved taxonomic resolution over 16S rRNA V4. Moreover, the results reveal that different ASVs within a species were found to change in abundance at different stages of caries progression. These findings suggest that strains of a single species may perform distinct roles along a biochemical spectrum associated with health and disease. The improved identification of oral streptococcal species will provide a better understanding of the different ecological roles of oral streptococci and inform the design of novel oral probiotic formulations for prevention and treatment of dental caries. IMPORTANCE The microbiota associated with the initiation and progression of dental caries has yet to be fully characterized. Although much insight has been gained from 16S rRNA hypervariable region DNA sequencing, this approach has several limitations, including poor taxonomic resolution at the species level. This is particularly relevant for oral streptococci, which are abundant members of oral biofilm communities and major players in health and caries disease. Here, we develop a new method for taxonomic profiling of oral streptococci based on the 30S-S11 rRNA gene, which provides much improved resolution over 16S rRNA V4 (resolving 10 as opposed to 2 species). Importantly, 30S-S11 can resolve multiple amplicon sequence variants (ASVs) within species, providing an unprecedented insight into the ecological progression of caries. For example, our findings reveal multiple incidences of different ASVs within a species with contrasting associations with health or disease, a finding that has high relevance toward the informed design of prebiotic and probiotic therapy.

Differences in the Accessory Genomes and Methylomes of Strains of Streptococcus equi subsp. equi and of Streptococcus equi subsp. zooepidemicus Obtained from the Respiratory Tract of Horses from Texas.

Streptococcus equi subsp. equi (SEE) is a host-restricted equine pathogen considered to have evolved from Streptococcus equi subsp. zooepidemicus (SEZ). SEZ is promiscuous in host range and is commonly recovered from horses as a commensal. Comparison of a single strain each of SEE and SEZ using whole-genome sequencing, supplemented by PCR of selected genes in additional SEE and SEZ strains, was used to characterize the evolution of SEE. But the known genetic variability of SEZ warrants comparison of the whole genomes of multiple SEE and SEZ strains. To fill this knowledge gap, we utilized whole-genome sequencing to characterize the accessory genome elements (AGEs; i.e., elements present in some SEE strains but absent in SEZ or vice versa) and methylomes of 50 SEE and 50 SEZ isolates from Texas. Consistent with previous findings, AGEs consistently found in all SEE isolates were primarily from mobile genetic elements that might contribute to host restriction or pathogenesis of SEE. Fewer AGEs were identified in SEZ because of the greater genomic variability among these isolates. The global methylation patterns of SEE isolates were more consistent than those of the SEZ isolates. Among homologous genes of SEE and SEZ, differential methylation was identified only in genes of SEE encoding proteins with functions of quorum sensing, exopeptidase activity, and transitional metal ion binding. Our results indicate that effects of genetic mobile elements in SEE and differential methylation of genes shared by SEE and SEZ might contribute to the host specificity of SEE. IMPORTANCE Strangles, caused by the host-specific bacterium Streptococcus equi subsp. equi (SEE), is the most commonly diagnosed infectious disease of horses worldwide. Its ancestor, Streptococcus equi subsp. zooepidemicus (SEZ), is frequently isolated from a wide array of hosts, including horses and humans. A comparison of the genomes of a single strain of SEE and SEZ has been reported, but sequencing of further isolates has revealed variability among SEZ strains. Thus, the importance of this study is that it characterizes genomic and methylomic differences of multiple SEE and SEZ isolates from a common geographic region (viz., Texas). Our results affirm many of the previously described differences between the genomes of SEE and SEZ, including the role of mobile genetic elements in contributing to host restriction. We also provide the first characterization of the global methylome of Streptococcus equi and evidence that differential methylation might contribute to the host restriction of SEE.

Streptococcus pneumoniae serotypes that frequently colonise the human nasopharynx are common recipients of penicillin-binding protein gene fragments from Streptococcus mitis.

Streptococcus pneumoniae is an important global pathogen that causes bacterial pneumonia, sepsis and meningitis. Beta-lactam antibiotics are the first-line treatment for pneumococcal disease, however, their effectiveness is hampered by beta-lactam resistance facilitated by horizontal genetic transfer (HGT) with closely related species. Although interspecies HGT is known to occur among the species of the genus Streptococcus, the rates and effects of HGT between Streptococcus pneumoniae and its close relatives involving the penicillin binding protein (pbp) genes remain poorly understood. Here we applied the fastGEAR tool to investigate interspecies HGT in pbp genes using a global collection of whole-genome sequences of Streptococcus mitis, Streptococcus oralis and S. pneumoniae. With these data, we established that pneumococcal serotypes 6A, 13, 14, 16F, 19A, 19F, 23F and 35B were the highest-ranking serotypes with acquired pbp fragments. S. mitis was a more frequent pneumococcal donor of pbp fragments and a source of higher pbp nucleotide diversity when compared with S. oralis. Pneumococci that acquired pbp fragments were associated with a higher minimum inhibitory concentration (MIC) for penicillin compared with pneumococci without acquired fragments. Together these data indicate that S. mitis contributes to reduced ß-lactam susceptibility among commonly carried pneumococcal serotypes that are associated with long carriage duration and high recombination frequencies. As pneumococcal vaccine programmes mature, placing increasing pressure on the pneumococcal population structure, it will be important to monitor the influence of antimicrobial resistance HGT from commensal streptococci such as S. mitis.

Identification of Streptococcus pseudopneumoniae and other mitis group streptococci using matrix assisted laser desorption/ionization - time of flight mass spectrometry.

This study evaluated the ability of the MALDI-ToF MS from Bruker Daltonics to identify clinical Mitis-Group-Streptococcus isolates with a focus on Streptococcus pseudopneumoniae. The results were analyzed using the standard log(score) and the previously published list(score). Importantly, using the log(score) no misidentifications occurred and 27 of 29 (93%) S. pneumoniae and 27 of 30 (90%) S. oralis strains were identified, but only 1 of 31 (3%) S. pseudopneumoniae and 1 of 13 (8%) S. mitis strains were identified. However, our results show that 30 of 31 S. pseudopneumoniae strains had a S. pseudopneumoniae Main Spectral Profiles within the 3 best matches. Using the list(score) all S. oralis and S. pneumoniae strains were identified correctly, but list(score) misidentified 10 S. pseudopneumoniae and 5 S. mitis. We propose to use the log(score) for identification of S. pneumoniae, S. pseudopneumoniae, S. mitis and S. oralis, but for some strains additional testing may be needed.

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.

Differences in the genome, methylome, and transcriptome do not differentiate isolates of Streptococcus equi subsp. equi from horses with acute clinical signs from isolates of inapparent carriers.

Streptococcus equi subsp. equi (SEE) is a host-restricted bacterium that causes the common infectious upper respiratory disease known as strangles in horses. Perpetuation of SEE infection appears attributable to inapparent carrier horses because it neither persists long-term in the environment nor infects other host mammals or vectors, and infection results in short-lived immunity. Whether pathogen factors enable SEE to remain in horses without causing clinical signs remains poorly understood. Thus, our objective was to use next-generation sequencing technologies to characterize the genome, methylome, and transcriptome of isolates of SEE from horses with acute clinical strangles and inapparent carrier horses-including isolates recovered from individual horses sampled repeatedly-to assess pathogen-associated changes that might reflect specific adaptions of SEE to the host that contribute to inapparent carriage. The accessory genome elements and methylome of SEE isolates from Sweden and Pennsylvania revealed no significant or consistent differences between acute clinical and inapparent carrier isolates of SEE. RNA sequencing of SEE isolates from Pennsylvania demonstrated no genes that were differentially expressed between acute clinical and inapparent carrier isolates of SEE. The absence of specific, consistent changes in the accessory genomes, methylomes, and transcriptomes of acute clinical and inapparent carrier isolates of SEE indicates that adaptations of SEE to the host are unlikely to explain the carrier state of SEE. Efforts to understand the carrier state of SEE should instead focus on host factors.

Exploring the signature gut and oral microbiome in individuals of specific Ayurveda prakriti.

Diagnosis and treatment of various diseases in Ayurveda, the Indian system of medicine, relies on 'prakriti' phenotyping of individuals into predominantly three constitutions, kapha, pitta and vata. Recent studies propose that microbiome play an integral role in precision medicine. A study of the relationship between prakriti - the basis of personalized medicine in Ayurveda and that of gut microbiome, and possible biomarker of an individual's health, would vastly improve precision therapy. Towards this, we analyzed bacterial metagenomes from buccal (oral microbiome) and fecal (gut microbiome) samples of 272 healthy individuals of various predominant prakritis. Major bacterial genera from gut microbiome included Prevotella, Bacteroides and Dialister while oral microbiome included Streptococcus, Neisseria, Veilonella, Haemophilus, Porphyromonas and Prevotella. Though the core microbiome was shared across all individuals, we found prakriti specific signatures such as preferential presence of Paraprevotella and Christensenellaceae in vata individuals. A comparison of core gut microbiome of each prakriti with a database of 'healthy' microbes identified microbes unique to each prakriti with functional roles similar to the physiological characteristics of various prakritis as described in Ayurveda. Our findings provide evidence to Ayurvedic interventions based on prakriti phenotyping and possible microbial biomarkers that can stratify the heterogenous population and aid in precision therapy.