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.

Genetic organization of an M protein trans-acting positive regulator (Mga) orthologue and its adjacent M-like protein (SCM) alleles in Streptococcus canis.

OBJECTIVE: The purpose of this study was to identify the M protein trans-acting positive regulator (Mga) orthologue and its adjacent M-like protein (SCM) alleles in Streptococcus canis. RESULTS: Using the 39 SCM allele isolates and polymerase chain reaction-based amplification and sequencing, we obtained the deduced Mga amino acid (AA) sequences. The 22 Mga sequences in whole-genome sequences were obtained by searching the National Collection of Type Cultures 12,191(T) Mga sequence into the database. The percentage identity to the type-strain Mga sequence was examined along with its size. The presence of the Mga-specific motifs was confirmed. Of the 62 strains, we identified 59 Mga sequences with an AA size of 509 (except for four different sizes). Percentage identity ranged from 96.66 to 100% with the confirmed Mga-specific motifs and diverse SCM allele populations. Our findings support the presence of an Mga orthologue and diverse SCM allele populations.

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).

Genome analysis of Streptococcus spp. isolates from animals in pre-antibiotic era with respect to antibiotic susceptibility and virulence gene profiles.

Lyophilized Streptococcus spp. isolates (n = 50) from animal samples submitted to the diagnostic laboratory at the University of Connecticut in the 1940s were revivified to investigate the genetic characteristics using whole-genome sequencing (WGS). The Streptococcus spp. isolates were identified as follows; S. agalactiae (n = 14), S. dysgalactiae subsp. dysgalactiae (n = 10), S. dysgalactiae subsp. equisimils (n = 5), S. uberis (n = 8), S. pyogenes (n = 7), S. equi subsp. zooepidemicus (n = 4), S. oralis (n = 1), and S. pseudoporcinus (n = 1). We identified sequence types (ST) of S. agalactiae, S. dysgalactiae, S. uberis, S. pyogenes, and S. equi subsp. zooepidemicus and reported ten novel sequence types of those species. WGS analysis revealed that none of Streptococcus spp. carried antibiotic resistance genes. However, tetracycline resistance was observed in four out of 15 S. dysgalactiae isolates and in one out of four S. equi subsp. zooepidemicus isolate. This data highlights that antimicrobial resistance is pre-existed in nature before the use of antibiotics. The draft genome sequences of isolates from this study and 426 complete genome sequences of Streptococcus spp. downloaded from BV-BRC and NCBI GenBank database were analyzed for virulence gene profiles and phylogenetic relationships. Different Streptococcus species demonstrated distinct virulence gene profiles, with no time-related variations observed. Phylogenetic analysis revealed high genetic diversity of Streptococcus spp. isolates from the 1940s, and no clear spatio-temporal clustering patterns were observed among Streptococcus spp. analyzed in this study. This study provides an invaluable resource for studying the evolutionary aspects of antibiotic resistance acquisition and virulence in Streptococcus spp.

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.

Overlapping Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis household transmission and mobile genetic element exchange.

Streptococcus dysgalactiae subspecies equisimilis (SDSE) and Streptococcus pyogenes share skin and throat niches with extensive genomic homology and horizontal gene transfer (HGT) possibly underlying shared disease phenotypes. It is unknown if cross-species transmission interaction occurs. Here, we conduct a genomic analysis of a longitudinal household survey in remote Australian First Nations communities for patterns of cross-species transmission interaction and HGT. Collected from 4547 person-consultations, we analyse 294 SDSE and 315 S. pyogenes genomes. We find SDSE and S. pyogenes transmission intersects extensively among households and show that patterns of co-occurrence and transmission links are consistent with independent transmission without inter-species interference. We identify at least one of three near-identical cross-species mobile genetic elements (MGEs) carrying antimicrobial resistance or streptodornase virulence genes in 55 (19%) SDSE and 23 (7%) S. pyogenes isolates. These findings demonstrate co-circulation of both pathogens and HGT in communities with a high burden of streptococcal disease, supporting a need to integrate SDSE and S. pyogenes surveillance and control efforts.

Antimicrobial resistance patterns of Streptococcus uberis isolates from bovine milk in Chiba prefecture, Japan: association between multidrug resistance and clonal complex 996.

Streptococcus uberis is one of major pathogens causing bovine mastitis. However, there is poor information on antimicrobial resistance (AMR) among the Japanese isolates. To provide treatment information for the mastitis caused by S. uberis in Japan, we aimed to clarify AMR patterns of the isolates from bovine milk mainly in Chiba. AMR phenotyping/genotyping [blaZ-erm(A)-erm(B)-mef(A)-linB-lnuD-tet(M)-tet(O)-tet(K)-tet(L)-tet(S)] and multilocus sequence typing were performed to analyze relationships between AMR patterns and clonal complexes (CCs). Resistance to tetracycline-, macrolide-, and lincosamide-classes was mainly associated with possession of tet(O), tet(S), erm(B), linB, and lnuD genes. CC996 was significantly associated with multidrug resistance (P<0.0001). These findings will aid Chiba farm animal clinics in treating bovine mastitis.

Comparative whole genome analysis of face-derived Streptococcus infantis CX-4 unravels the functions related to skin barrier.

BACKGROUND: The skin microbiome is essential in guarding against harmful pathogens and responding to environmental changes by generating substances useful in the cosmetic and pharmaceutical industries. Among these microorganisms, Streptococcus is a bacterial species identified in various isolation sources. In 2021, a strain of Streptococcus infantis, CX-4, was identified from facial skin and found to be linked to skin structure and elasticity. As the skin-derived strain differs from other S. infantis strains, which are usually of oral origin, it emphasizes the significance of bacterial variation by the environment. OBJECTIVE: This study aims to explore the unique characteristics of the CX-4 compared to seven oral-derived Streptococcus strains based on the Whole-Genome Sequencing data, focusing on its potential role in skin health and its possible application in cosmetic strategies. METHODS: The genome of the CX-4 strain was constructed using PacBio Sequencing, with the assembly performed using the SMRT protocol. Comparative whole-genome analysis was then performed with seven closely related strains, utilizing web-based tools like PATRIC, OrthoVenn3, and EggNOG-mapper, for various analyses, including protein association analysis using STRING. RESULTS: Our analysis unveiled a substantial number of Clusters of Orthologous Groups in diverse functional categories in CX-4, among which sphingosine kinase (SphK) emerged as a unique product, exclusively present in the CX-4 strain. SphK is a critical enzyme in the sphingolipid metabolic pathway, generating sphingosine-1-phosphate. The study also brought potential associations with isoprene formation and retinoic acid synthesis, the latter being a metabolite of vitamin A, renowned for its crucial function in promoting skin cell growth, differentiation, and maintaining of skin barrier integrity. These findings collectively suggest the potential of the CX-4 strain in enhancing of skin barrier functionality. CONCLUSION: Our research underscores the potential of the skin-derived S. infantis CX-4 strain by revealing unique bacterial compounds and their potential roles on human skin.

Identification and genetic engineering of pneumococcal capsule-like polysaccharides in commensal oral streptococci.

Capsular polysaccharides (CPS) in Streptococcus pneumoniae are pivotal for bacterial virulence and present extensive diversity. While oral streptococci show pronounced antigenicity toward pneumococcal capsule-specific sera, insights into evolution of capsule diversity remain limited. This study reports a pneumococcal CPS-like genetic locus in Streptococcus parasanguinis, a predominant oral Streptococcus. The discovered locus comprises 15 genes, mirroring high similarity to those from the Wzy-dependent CPS locus of S. pneumoniae. Notably, S. parasanguinis elicited a reaction with pneumococcal 19B antiserum. Through nuclear magnetic resonance analysis, we ascertained that its CPS structure matches the chemical composition of the pneumococcal 19B capsule. By introducing the glucosyltransferase gene cps19cS from a pneumococcal serotype 19C, we successfully transformed S. parasanguinis antigenicity from 19B to 19C. Furthermore, substituting serotype-specific genes, cpsI and cpsJ, with their counterparts from pneumococcal serotype 19A and 19F enabled S. parasanguinis to generate 19A- and 19F-specific CPS, respectively. These findings underscore that S. parasanguinis harbors a versatile 19B-like CPS adaptable to other serotypes. Remarkably, after deleting the locus's initial gene, cpsE, responsible for sugar transfer, we noted halted CPS production, elongated bacterial chains, and diminished biofilm formation. A similar phenotype emerged with the removal of the distinct gene cpsZ, which encodes a putative autolysin. These data highlight the importance of S. parasanguinis CPS for biofilm formation and propose a potential shared ancestry of its CPS locus with S. pneumoniae. IMPORTANCE: Diverse capsules from Streptococcus pneumoniae are vital for bacterial virulence and pathogenesis. Oral streptococci show strong responses to a wide range of pneumococcal capsule-specific sera. Yet, the evolution of this capsule diversity in relation to microbe-host interactions remains underexplored. Our research delves into the connection between commensal oral streptococcal and pneumococcal capsules, highlighting the potential for gene transfer and evolution of various capsule types. Understanding the genetic and evolutionary factors that drive capsule diversity in S. pneumoniae and its related oral species is essential for the development of effective pneumococcal vaccines. The present findings provide fresh perspectives on the cross-reactivity between commensal streptococci and S. pneumoniae, its influence on bacteria-host interactions, and the development of new strategies to manage and prevent pneumococcal illnesses by targeting and modulating commensal streptococci.

Expanding the flexibility of base editing for high-throughput genetic screens in bacteria.

Genome-wide screens have become powerful tools for elucidating genotype-to-phenotype relationships in bacteria. Of the varying techniques to achieve knockout and knockdown, CRISPR base editors are emerging as promising options. However, the limited number of available, efficient target sites hampers their use for high-throughput screening. Here, we make multiple advances to enable flexible base editing as part of high-throughput genetic screening in bacteria. We first co-opt the Streptococcus canis Cas9 that exhibits more flexible protospacer-adjacent motif recognition than the traditional Streptococcus pyogenes Cas9. We then expand beyond introducing premature stop codons by mutating start codons. Next, we derive guide design rules by applying machine learning to an essentiality screen conducted in Escherichia coli. Finally, we rescue poorly edited sites by combining base editing with Cas9-induced cleavage of unedited cells, thereby enriching for intended edits. The efficiency of this dual system was validated through a conditional essentiality screen based on growth in minimal media. Overall, expanding the scope of genome-wide knockout screens with base editors could further facilitate the investigation of new gene functions and interactions in bacteria.

A core genome multi-locus sequence typing scheme for Streptococcus uberis: an evolution in typing a genetically diverse pathogen.

Streptococcus uberis is a globally endemic and poorly controlled cause of bovine mastitis impacting the sustainability of the modern dairy industry. A core genome was derived from 579 newly sequenced S. uberis isolates, along with 305 publicly available genome sequences of S. uberis isolated from 11 countries around the world and used to develop a core genome multi-locus sequence typing (cgMLST) scheme. The S. uberis core genome comprised 1475 genes, and these were used to identify 1447 curated loci that were indexed into the cgMLST scheme. This was able to type 1012 of 1037 (>97  %) isolates used and differentiated the associated sequences into 932 discrete core genome sequence types (cgSTs). Analysis of the phylogenetic relationships of cgSTs revealed no clear clustering of isolates based on metadata such as disease status or year of isolation. Geographical clustering of cgSTs was limited to identification of a UK-centric clade, but cgSTs from UK isolates were also dispersed with those originating from other geographical regions across the entire phylogenetic topology. The cgMLST scheme offers a new tool for the detailed analysis of this globally important pathogen of dairy cattle. Initial analysis has re-emphasized and exemplified the genetically diverse nature of the global population of this opportunistic pathogen.

Streptococcus taonis sp. nov., a novel bacterial species isolated from a blood culture of a patient.

One Gram stain-positive, catalase-negative, α-hemolytic, chain-forming or paired cocci, designated ST22-14T, was isolated from a blood culture of a child with suspected infection. The results of 16S rRNA gene sequences analyses showed that the most closely related species to strain ST22-14T were "Streptococcus vulneris" DM3B3T (99.2%), Streptococcus mitis NCTC 12261T (99.0%), "Streptococcus gwangjuense" ChDC B345T, (99.0%), Streptococcus oralis subsp. dentisani 7747T (99.0%), Streptococcus downii CECT 9732T (99.0%), and Streptococcus infantis ATCC 700779T (98.9%). The genome of strain ST22-14T consists of 2,053,261 bp with a G + C content of 39.4%. Average nucleotide identity values between strain ST22-14T and Streptococcus mitis NCTC 12261T or other five species were from 82.2 to 88.0%. In silico DNA-DNA hybridization of ST22-14T showed an estimated DNA reassociation value of 34.6% with the closest species. The main cellular fatty acids of strain ST22-14T were 16:0, 18:0, 14:0, 18:1ω7c and 18:1ω6c. Based on these results, strain ST22-14T should be classified as a novel species of genus Streptococcus, for which the name Streptococcus taonis sp. nov. is proposed (type strain ST22-14T = NBRC 116002T = BCRC 81402T).

Human saliva modifies growth, biofilm architecture, and competitive behaviors of oral streptococci.

The bacteria within supragingival biofilms participate in complex exchanges with other microbes inhabiting the same niche. One example is the mutans group streptococci (Streptococcus mutans), implicated in the development of tooth decay, and other health-associated commensal streptococci species. Previously, our group transcriptomically characterized intermicrobial interactions between S. mutans and several species of oral bacteria. However, these experiments were carried out in a medium without human saliva. To better mimic their natural environment, we first evaluated how inclusion of saliva affected growth and biofilm formation of eight Streptococcus species individually and found saliva to positively benefit growth rates while negatively influencing biofilm biomass accumulation and altering spatial arrangement. These results carried over during evaluation of 29 saliva-derived isolates of various species. Surprisingly, we also found that addition of saliva increased the competitive behaviors of S. mutans in coculture competitions against commensal streptococci that led to increases in biofilm microcolony volumes. Through transcriptomically characterizing mono- and cocultures of S. mutans and Streptococcus oralis with and without saliva, we determined that each species developed a nutritional niche under mixed-species growth, with S. mutans upregulating carbohydrate uptake and utilization pathways while S. oralis upregulated genome features related to peptide uptake and glycan foraging. S. mutans also upregulated genes involved in oxidative stress tolerance, particularly manganese uptake, which we could artificially manipulate by supplementing in manganese leading to an advantage over its opponent. Our report highlights observable changes in microbial behaviors through leveraging environmental- and host-supplied resources over their competitors. IMPORTANCE: Dental caries (tooth decay) is the most prevalent disease for both children and adults nationwide. Caries are initiated from demineralization of the enamel due to organic acid production through the metabolic activity of oral bacteria growing in biofilm communities attached to the tooth's surface. Mutans group streptococci are closely associated with caries development and initiation of the cariogenic cycle, which decreases the amount of acid-sensitive, health-associated commensal bacteria while selecting for aciduric and acidogenic species that then further drives the disease process. Defining the exchanges that occur between mutans group streptococci and oral commensals in a condition that closely mimics their natural environment is of critical need toward identifying factors that can influence odontopathogen establishment, persistence, and outgrowth. The goal of our research is to develop strategies, potentially through manipulation of microbial interactions characterized here, that prevent the emergence of mutans group streptococci while keeping the protective flora intact.

Exploring nasopharyngeal microbiota profile in children affected by SARS-CoV-2 infection.

The relationship between COVID-19 and nasopharyngeal (NP) microbiota has been investigated mainly in the adult population. We explored the NP profile of children affected by COVID-19, compared to healthy controls (CTRLs). NP swabs of children with COVID-19, collected between March and September 2020, were investigated at the admission (T0), 72 h to 7 days (T1), and at the discharge (T2) of the patients. NP microbiota was analyzed by 16S rRNA targeted-metagenomics. Data from sequencing were investigated by QIIME 2.0 and PICRUSt 2. Multiple machine learning (ML) models were exploited to classify patients compared to CTRLs. The NP microbiota of COVID-19 patients (N = 71) was characterized by reduction of α-diversity compared to CTRLs (N = 59). The NP microbiota of COVID-19 cohort appeared significantly enriched in Streptococcus, Haemophilus, Staphylococcus, Veillonella, Enterococcus, Neisseria, Moraxella, Enterobacteriaceae, Gemella, Bacillus, and reduced in Faecalibacterium, Akkermansia, Blautia, Bifidobacterium, Ruminococcus, and Bacteroides, compared to CTRLs (FDR < 0.001). Exploiting ML models, Enterococcus, Pseudomonas, Streptococcus, Capnocytopagha, Tepidiphilus, Porphyromonas, Staphylococcus, and Veillonella resulted as NP microbiota biomarkers, in COVID-19 patients. No statistically significant differences were found comparing the NP microbiota profile of COVID-19 patients during the time-points or grouping patients on the basis of high, medium, and low viral load (VL). This evidence provides specific pathobiont signatures of the NP microbiota in pediatric COVID-19 patients, and the reduction of anaerobic protective commensals. Our data suggest that the NP microbiota may have a specific disease-related signature since infection onset without changes during disease progression, regardless of the SARS-CoV-2 VL. IMPORTANCE: Since the beginning of pandemic, we know that children are less susceptible to severe COVID-19 disease. A potential role of the nasopharyngeal (NP) microbiota has been hypothesized but to date, most of the studies have been focused on adults. We studied the NP microbiota modifications in children affected by SARS-CoV-2 infection showing a specific NP microbiome profile, mainly composed by pathobionts and almost missing protective anaerobic commensals. Moreover, in our study, specific microbial signatures appear since the first days of infection independently from SARS-CoV-2 viral load.

Analysis of subgingival bacterial and fungal diversity in patients with peri-implantitis based on 16sRNA and internal transcribed spacer sequencing.

Aim: To analyze subgingival fungal diversity in peri-implant inflammation patients and their relationship with bacteria. Methods: We collected saliva samples from four groups. 16sRNA and internal transcribed spacer sequencing was performed preceded by quantitative PCR and enzyme-linked immunosorbent assay tests. Analyses were done using R and Cytoscape software. Results: Significant differences were observed in the Abundance-based Coverage Estimator (ACE) index between control and peri-implantitis samples. Basidiomycota was the dominant fungal species, while Firmicutes dominated the bacteria. The most abundant fungal and bacterial species were 's_unclassified g Apiotrichum' and 's_unclassified g Streptococcus', respectively. Dothiorella was strongly associated with immunoglobulin G levels, with positive correlations between specific microorganisms and peri-implantitis in Q-PCR. Conclusion: Our findings have significant clinical implications, suggesting specific fungal and bacterial taxa roles in peri-implant inflammation.

Streptococcus ruminantium-associated sheep mastitis outbreak detected in Italy is distinct from bovine isolates.

Streptococcus ruminantium is the causative agent of several bovine and ovine diseases, however reports are uncommon and application of whole genome sequencing to identify is rare. We report for the first time, a severe ovine mastitis outbreak caused by S. ruminantium in Italy, 2022. S. ruminantium was isolated from 12 adult lactating ewes with diffuse nodules in the mammary parenchyma and predominantly serous and clotted milk. All outbreak isolates, along with five additional historical Italian isolates (between 2011 and 2017), were genomically characterised and then analysed in the context of all publicly available S. ruminantium genomes. Antimicrobial susceptibility testing was performed to determine the MICs of 16 antibiotics. The results showed that all isolates were susceptible to all antimicrobials tested except kanamycin. Single Nucleotide Variant analysis confirmed this as a clonal outbreak across 10 sheep (≤ 15 SNVs), while the two others were colonised by more distantly related clones (≤ 53 pairwise SNVs), indicating the presence of multiple infecting lineages. The five historical S. ruminantium isolates were comprised of genetically-distant singletons (between 1259 and 5430 pairwise SNVs to 2022 outbreak isolates). Ovine isolates were found to be genetically distinct to bovine isolates, forming monophyletic groups. Bovine isolates were similarly made up of singleton clones in all but two isolates. Taken together, our genomic analysis using all globally available genomes is consistent with general opportunistic pathogenesis of S. ruminantium. We encourage future genomic surveillance efforts to facilitate outbreak detection, as well as improve our understanding of this poorly-understood, multi-host, zoonotic pathogen.

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.

Accurate identification of Streptococcus pseudopneumoniae and other mitis group Streptococci identified as atypical Streptococcus pneumoniae in Tunisian pediatric population.

Accurate identification of Mitis group streptococci especially Streptococcus pneumoniae and Streptococcus pseudopneumoniae seems difficult due to the lack of specific and sensitive tests. We performed an approach for the identification of atypical pneumococci in pediatric Tunisian population. In this study, 49 streptococcal isolates that were considered as atypical S. pneumoniae were analyzed by: optochin susceptibility in ambient and 5% CO2 atmosphere, oxgall disk sensitivity, PCR targeting several genes and antimicrobial susceptibility.The combined results of biochemical and molecular methods showed the presence of 23 S. pneumoniae, 7 S. pseudopneumoniae, and 19 other mitis group. Among S. pseudopneumoniae, all isolates were collected from respiratory tract samples and showed a high level of resistance to ß-lactams with a MIC90 of 32 mg L-1. Two isolates of S. pseudopneumoniae showed the typical phenotype of optochin resistance described in the literature. All isolates could be identified only by molecular tests. Among Streptococcus pneumonaie, all strains harbored the lytA gene and the Spn9802 fragment. But only 14 strains were encapsulated.This study describes several assays for the identification of atypical pneumococci in order to gain insights on the nature of isolate and raise alert about the presence of these strains in the pediatric Tunisian community.

Comparative genomics analysis of Streptococcus iniae isolated from Trachinotus ovatus: novel insight into antimicrobial resistance and virulence differentiation.

BACKGROUND: Streptococcus iniae is an important fish pathogen that cause significant economic losses to the global aquaculture industry every year. Although there have some reports on the genotype of S.iniae and its relationship with virulence, no genome-scale comparative analysis has been performed so far. In our previous work, we characterized 17 isolates of S.iniae from Trachinotus ovatus and divided them into two genotypes using RAPD and rep-PCR methods. Among them, BH15-2 was classified as designated genotype A (in RAPD) and genotype 1 (in rep-PCR), while BH16-24 was classified as genotype B and genotype 2. Herein, we compared the differences in growth, drug resistance, virulence, and genome between BH15-2 and BH16-24. RESULTS: The results showed that the growth ability of BH16-24 was significantly faster than that of BH15-2 at the exponential stage. Antimicrobial tests revealed that BH15-2 was susceptible to most of the tested antibiotics except neomycin and gentamycin. In contrast, BH16-24 was resistant to 7 antibiotics including penicillin, sulfasomizole, compound sulfamethoxazole tablets, polymyxin B, spectinomycin, rifampin and ceftazidime. Intraperitoneal challenge of T.ovatus, showed that the LD50 value of BH15-2 was 4.0 × 102 CFU/g, while that of BH16-24 was 1.2 × 105 CFU/g. The genome of S.iniae BH15-2 was 2,175,659 bp with a GC content of 36.80%. Meanwhile, the genome of BH16-24 was 2,153,918 bp with a GC content of 36.83%. Comparative genome analysis indicated that compared with BH15-2, BH16-24 genome had a large-scale genomic inversion fragment, at the location from 502,513 bp to 1,788,813 bp, resulting in many of virulence and resistance genes differentially expression. In addition, there was a 46 kb length, intact phage sequence in BH15-2 genome, which was absent in BH16-24. CONCLUSION: Comparative genomic studies of BH15-2 and BH16-24 showed that the main difference is a 1.28 Mbp inversion fragment. The inversion fragment may lead to abnormal expression of drug resistant and virulence genes, which is believed to be the main reason for the multiple resistance and weakened virulence of BH16-24. Our study revealed the potential mechanisms in underlying the differences of multidrug resistance and virulence among different genotypes of S.iniae.

Cell wall polysaccharides of streptococci: A genetic and structural perspective.

The Streptococcus genus comprises both commensal and pathogenic species. Additionally, Streptococcus thermophilus is exploited in fermented foods and in probiotic preparations. The ecological and metabolic diversity of members of this genus is matched by the complex range of cell wall polysaccharides that they present on their cell surfaces. These glycopolymers facilitate their interactions and environmental adaptation. Here, current knowledge on the genetic and compositional diversity of streptococcal cell wall polysaccharides including rhamnose-glucose polysaccharides, exopolysaccharides and teichoic acids is discussed. Furthermore, the species-specific cell wall polysaccharide combinations and specifically highlighting the presence of rhamnose-glucose polysaccharides in certain species, which are replaced by teichoic acids in other species. This review highlights model pathogenic and non-pathogenic species for which there is considerable information regarding cell wall polysaccharide composition, structure and genetic information. These serve as foundations to predict and focus research efforts in other streptococcal species for which such data currently does not exist.