Detailed structural characterization of five water-insoluble α-glucans produced by glucansucrases from Streptococcus spp.

Water-insoluble α-glucans synthesized from sucrose by glucansucrases from Streptococcus spp. are essential in dental plaque and caries formation. Because limited information is available on the fine structure of these biopolymers, we analyzed the structures of unmodified glucans produced by five recombinant Streptococcus (S.) mutans DSM 20523 and S. salivarius DSM 20560 glucansucrases in detail. A combination of methylation analysis, endo-dextranase and endo-mutanase hydrolyses, and HPSEC-RI was used. Furthermore, crystal-like regions were analyzed by using XRD and 13C MAS NMR spectroscopy. Our results showed that the glucan structures were highly diverse: Two glucans with 1,3- and 1,6-linkages were characterized in detail besides an almost exclusively 1,3-linked and a linear 1,6-linked glucan. Furthermore, one glucan contained 1,3-, 1,4-, and 1,6-linkages and thus had an unusual, not yet described structure. It was demonstrated that the glucans had a varying structural architecture by using partial enzymatic hydrolyses. Furthermore, crystal-like regions formed by 1,3-glucopyranose units were observed for the two 1,3- and 1,6-linked glucans and the linear 1,3-linked glucan. 1,6-linked regions were mobile and not involved in the crystal-like areas. Altogether, our results broaden the knowledge of the structure of water-insoluble α-glucans from Streptococcus spp.

Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

Development of novel Streptococcus equi vaccines with an assessment of their immunizing potentials and protective efficacies.

Strangles is a highly contagious disease of the equine upper respiratory tract caused by Streptococcus equi subspecies. Streptococcus equi subsp. equi (S. equi) and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) was isolated, as local, hot, and field strains, from horses clinically suffering from respiratory distress. The isolated Streptococci were identified using bacteriological and molecular techniques. Four formulations of inactivated S. equi vaccines were developed and evaluated. The first formulation was prepared using the S. equi isolates, adjuvanted with MONTANIDE GEL adjuvant, while the second formulation was adjuvanted with MONTANIDE ISA-70 adjuvant. The other 2 formulations were inactivated combined vaccines prepared from both S. equi and S. zooepidemicus isolates. The 3rd formulation was the combined isolates adjuvanted with MONTANIDE GEL while the 4th formulation was the combined isolates adjuvanted with MONTANIDE ISA-70. The developed vaccines' physical properties, purity, sterility, safety, and potency were ensured. The immunizing efficacy was determined in isogenic BALB/c mice and white New Zealand rabbits using the passive hemagglutination test. Also, the antibodies' titer of the combined S. equi and S. zooepidemicus vaccine adjuvanted with MONTANIDE ISA-70 in foals was tracked using an indirect enzyme-linked immunosorbent assay. The protective efficacy of the developed vaccines was determined using a challenge test in both laboratory and field animal models, where a 75% protection rate was achieved. The combined vaccine proved to be more efficacious than the monovalent vaccine. Also, the MONTANIDE ISA-70 adjuvant provided significant protective efficacy than the MONTANIDE GEL. The current work is introducing a very promising mitigative and strategic controlling solution for strangles.

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).

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 gallolyticus Subspecies (subsp.) pasteurianus Meningitis in a 7-week-old Boy.

Meningitis caused by Streptococcus gallolyticus subspecies (subsp.) pasteurianus is a rare complication with 14 cases reported in literature worldwide between 2003-2023, with the majority of the cases occurring before 4 weeks of life and with preceding symptoms. This is a case report of an infection without any preceding symptoms. A previously healthy 7-week-old boy presented to the hospital with a fever for 1 day. Blood and cerebrospinal fluid cultures ultimately grew Streptococcus gallolyticus subsp. pasteurianus. The magnetic resonance imaging was consistent with meningitis. The boy received 21 days of intravenous antibiotics before discharge. At subsequent visits, the boy had no neurological sequelae, normal hearing tests, and appeared to have met all developmental milestones. The older age of infant should not discount the differential diagnosis for meningitis, which may delay further work up such as a lumbar puncture. Group D streptococcus is an uncommon cause of infantile sepsis that can lead to several complications such as meningitis and bacteremia. In this case, the infant's subsequent post-meningitis clinical course has been unremarkable. The history of meningitis poses increased risk for abnormal neurodevelopmental outcome. This case study highlights the importance of keeping meningitis on the differential diagnosis for an infant with fever. If there is a concern for meningitis, further workup should be performed without delay.

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.

Summer mortality syndrome bacterial pathogens in farmed Nile tilapia (Oreochromis niloticus).

Background: The high summer mortality in many fish farms, which had detrimental economic and social implications, was a serious challenge that the fish industry had to deal with. Aim: With an examination of the most effective antibiotic, the ongoing research was intended to shed light on the identification of the main bacterial pathogens associated with the summer mortality syndrome in the diseased farmed Nile tilapia. Methods: Six hundred dead Nile tilapia samples that had suffered from summer mortality were collected from several fish farms between May and October of 2022. The gathered fish displayed hemorrhagic areas on the skin, scale detachment, fin degeneration, erosions, skin ulcers, and corneal opacity with unilateral and/or bilateral exophthalmia. The most prominent internal appearance was swelling of the internal organs with sanguineous ascetic fluid. Results: There were 225 bacterial isolates found. Six species were identified through phenotypic and biochemical analysis; they were Aeromonas, Vibrio, Streptococcus, Pseudomonas, Enterococcus, and Edwardsiella spp., in descending percentage, respectively. Aeromonas spp., Vibrio spp., and Streptococcus spp. were the three most frequent isolated bacterial pathogens. The identification of Aeromonas hydrophila, Vibrio spp., and Streptococcus iniae, the three most common bacterial isolates, was confirmed by molecular analysis by polymerase chain reaction. Most of the tested strains were found to be responsive to Ciprofloxacin (CIP), Gentamicin (CN), and Chloramphenicol (C) but resistant to Amoxicillin (AMX), according to an antibiotic sensitivity test. Conclusion: The three most dangerous common bacterial infections discovered during mass-farmed tilapia summer mortality are A. hydrophil a, Vibrio sp., and S. iniae. This makes it clear that high water temperatures may raise the possibility of bacterial infections, which could cause widespread tilapia mortality and substantial financial losses. Therefore, it is crucial to maintain a beneficial fish culture, environment, and husbandry practices to enhance the tilapia-rearing environment and lessen the virulence of the disease. Isolated bacterial strains showed low levels of resistance to AMX but were vulnerable to CIP, CN, and C.

The discovery of an anti-inflammatory monoterpenoid, neoroseoside from the Zea mays.

A new monoterpenoid, neoroseoside (1), along with two previously reported compounds, 2″-O-α-l-rhamnosyl-6-C-fucosylluteolin (2) and farobin A (3) were isolated from the Zea mays. The structure of compound 1 was determined through the analysis spectroscopic data, including mass spectrometry (MS), infrared (IR) spectroscopy, and nuclear magnetic resonance (NMR) data. The absolute configurations of 1 were deduced from the comparing the values of optical rotations and from the interpretation of electronic circular dichroism (ECD) spectra. Compounds 2 and 3 displayed moderate antibacterial activity against Streptococcus mutans ATCC 25175 (inhibition rates 24 % and 28 %, respectively) and Streptococcus sobrinus ATCC 33478 (inhibition rate of 26 %), at a concentration of 100 µg/mL, whereas compound 1 did not have any significant antibacterial activities. The compounds 1-3 also showed anti-inflammatory activity on cytokine IL-6 and TNF-α.

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.

The antioxidant activity and metabolomic analysis of the supernatant of Streptococcus alactolyticus strain FGM.

Strain-specific probiotics can present antioxidant activity and reduce damage caused by oxidation. Streptococcus alactolyticus strain FGM (S. alactolyticus strain FGM) isolated from the chicken cecum shows potential probiotic properties which have been previously demonstrated. However, the antioxidant properties of S. alactolyticus strain FGM remain unknown. In this view, cell-free supernatant (CFS), intact cells (IC) and intracellular extracts (CFE) of strain FGM and 3 strains of Lactobacillus (LAB) were prepared, and their scavenging capacities against DPPH, hydroxyl radicals and linoleic acid peroxidation inhibitory were compared in this study. The effects of strain FGM cell-free supernatant (FCFS) on NO production, activity of SOD and GSH-Px in RAW264.7 cells and LPS-induced RAW264.7 cells were analyzed. The metabolites in the supernatant were quantitated by N300 Quantitative Metabolome. It was shown that the physicochemical characteristics of CFS to scavenge DPPH, hydroxyl radicals, and linoleic acid peroxidation inhibitory were significantly stronger than that of IC and CFE in the strain FGM (P < 0.05), respectively 87.12% ± 1.62, 45.03% ± 1.27, 15.63% ± 1.34. FCFS had a promotional effect on RAW264.7 cells, and significantly elevated SOD and GSH-Px activities in RAW264.7 cells. 25 µL FCFS significantly promoted the proliferation of RAW264.7 cells induced by LPS, increased the activities of SOD and GSH-PX, and decreased the release of NO. Furthermore, among the differential metabolites of FCFS quantified by N300, 12 metabolites were significantly up-regulated, including lactic acid, indole lactic acid, linoleic acid, pyruvic acid etc., many of which are known with antioxidant properties. In conclusion, FCFS had good antioxidant properties and activity, which can be attributed to metabolites produced from strain FGM fermentation. It was further confirmed that S. alactolyticus strain FGM and its postbiotic have potential probiotic properties and bright application prospects in livestock and poultry breeding.

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 in Patients with Vaginal Discharge: A 2019-2022 Analysis at the National Health Laboratory in Eritrea.

Background: Antimicrobial resistance poses a significant global threat to the treatment of bacterial infections, particularly in low- and middle-income regions such as Africa. This study is aimed at analyzing antimicrobial resistance patterns in vaginal swab samples from patients at the National Health Laboratory from 2019 to 2022. Methods: This retrospective study examined patient records from vaginal swab analyses performed at the National Health Laboratory between January 1, 2019, and December 31, 2022. Ethical approval was obtained from the Ministry of Health Research Ethical Approval and Clearance Committee on 15/02/2023. Results: Of the 622 samples, 83% underwent microbial isolation and identification. Citrobacter spp. exhibited high resistance (>43%) to antibiotics such as cephalexin, ceftazidime, nalidixic acid, ampicillin, gentamicin, and tetracycline. E. coli showed resistance rates of more than 50% to ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. Klebsiella spp. and Proteus spp. exhibited resistance rates that exceeded 47% to specific antibiotics. Gram-positive bacteria have resistance rates of more than 49% with ampicillin, trimethoprim-sulfamethoxazole, tetracycline, oxacillin, vancomycin, and penicillin G. In particular, S. aureus demonstrated no resistance to rifampicin or clindamycin, while Streptococcus spp. showed 100% resistance to rifampicin and vancomycin. Several species, including Proteus species, Streptococcus spp., S. aureus, and Klebsiella spp. exhibited multidrug resistance. Conclusion: Most gram-negative bacteria displayed higher resistance of >45% to ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. Among gram-positive bacteria, a higher resistance rate with ampicillin, trimethoprim-sulfamethoxazole, tetracycline, oxacillin, vancomycin, and penicillin G was recorded. S. aureus showed no resistance to rifampicin and clindamycin, and Strep. spp. indicated 100% resistance to rifampicin and vancomycin. This study highlights critical gaps and areas for further exploration. Expanding the spectrum of antibiotics tested and investigating underlying multidrug resistance mechanisms would provide a more comprehensive understanding of resistance patterns.

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.

Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

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.

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.

Isolation of phages against Streptococcus species in the oral cavity for potential control of dental diseases and associated systemic complications.

Dental infections and systemic complications caused by Streptococcus species in the oral cavity are increasingly exhibiting resistance to commonly used antibiotics, posing a potential threat to global public health. Phage therapy may offer a superior alternative, given that bacteriophages can be easily isolated and rapidly replicate in large numbers. In this study, six Streptococcus species from the oral cavity were characterized. Bacteriophages isolated from wastewater using five of these species as hosts produced plaques ranging from 0.2 to 2.4 mm in size. The phages demonstrated stability within a temperature range of 4 â„ƒ to 37 â„ƒ. However, at temperatures exceeding 45 â„ƒ, a noticeable reduction in bacteriophage titer was observed. Similarly, the phages showed greater stability within a pH range of 5 to 10. The isolated phages exhibited latency periods ranging from 15 to 20 min and had burst sizes varying from 10 to 200 viral particles. This study supports the potential use of bacteriophages in controlling infections caused by Streptococcus species.

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).