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Background: Hyaluronic acid (HA) is extensively employed in various fields such as medicine, cosmetics, food, etc. The molecular weight (MW) of HA is crucial for its biological functions. Streptococcus zooepidemicus, a prominent HA industrial producer, naturally synthetizes HA with high MW. Currently, few effective approaches exist for the direct and precise regulation of HA MW through a one-step fermentation process, and S. zooepidemicus lacks metabolic regulatory elements with varying intensities. The ratio of HA's precursors, UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA), is critical for the extension and release of HA. An imbalance in the precursor proportions for HA synthesis leads to a significant decrease in HA MW, indicating that controlling the precursor ratio may serve as a potential method for regulating HA MW. Results: In this study, the type and concentration of carbon sources were manipulated to disrupt the balance of precursor supply. Based on the results, it was speculated that the transcription level of hasE, which may connect the two HA synthesis precursors, is positively correlated with HA MW. Consequently, an endogenous expression component library for S. zooepidemicus was constructed, comprising 32 constitutive and 4 inducible expression elements. The expression of hasE was subsequently regulated in strain SE0 (S12 ΔhasE) using two constitutive promoters of differing strengths. The recombinant strain SE1, in which hasE was controlled by the stronger promoter PR31, produced HA with a MW of 1.96 MDa. In contrast, SE2, utilizing the weaker promoter PR22, synthesized shorter HA with a MW of 1.63 MDa, thereby verifying the hypothesis. Finally, to precisely regulate HA MW according to specific demands, an efficient sucrose-induced expression system was screened and employed to control the transcription level of hasE, obtaining recombinant strain SE3. When induced with sucrose concentrations of 3, 5–10 g/L, the HA MW of SE3 reached 0.78 to 1.77 MDa, respectively. Conclusions: Studies on regulating the balance of the HA precursor substances indicate that an oversupply of either UDP-GlcNAc or UDP-GlcUA can reduce HA MW. The hasE gene serves as a crucial regulator for maintaining this balance. Precise regulation of hasE transcription was achieved through an efficient inducible expression system, enabling the customized production of HA with specific MW. The HA MW of strain SE3 can be accurately manipulated by adjusting sucrose concentration, establishing a novel strategy for customized HA fermentation. [ABSTRACT FROM AUTHOR]

【Objective】The objective of this study is to explore the influencing elements of hyaluronic acid (HA) molecular weight, and to construct Streptococcus zooepidemicus mutants that synthesize HA with different molecular weights.【Method】When Streptococcus zooepidemicus S12 was cultured using different carbon sources to produce HA, it was found that the molecular weight of HA fermented by 10 g/Lfructose as the carbon source was 1.48×106 Da, which was 29.52% lower than that of the original fermentation medium (50 g/L sucrose as the carbon source, 2.10×106 Da) . Subsequently, transcriptomic sequencing of S12 fermentation broth with 50 g/L sucrose and 10 g/L fructose as carbon sources showed that the transcription levels of the two key genes of arginine deiminase pathway, arcA (encoding arginine deiminase) and argF (encoding ornithine carbamyltransferase), increased by 16.29 and 11.27 times, respectively, in addition to the fructose metabolism-related genes. In order to explore the effects of these two genes on HA synthesis, the genes arcA and argF were knocked out or overexpressed in S12, respectively.【Result】The HA molecular weight of arcA overexpressing strains was 2.96×106 Da in CDM medium, which was 50.25% higher than that of S12 (1.97×106 Da), and the molecular weight of HA in the other three mutant strains did not show conspicuous changes. Further RT-qPCR showed that the transcription level of glnA, the synthase-encoding gene of glutamine (amino donor in HA synthesis), was up-regulated by 2.0 times in arcA overexpressing strain, which may be one of the reasons for the increase of the HA molecular weight.【Conclusion】Two genes related to arginine metabolism were found to regulate the molecular weight of HA, which provides new targets for other strains to control the molecular weight of HA. [ABSTRACT FROM AUTHOR]

Hyaluronic acid (HA) is widely used in food and pharmaceutical industries. As a UDP-glucose dehydrogenase, hasB catalyzes the conversion of UDP-Glc to UDP-GlcUA in HA biosynthesis. However, little is known about structural characteristic and enzymatic property of hasB. Owing to high-yield HA production by Streptococcus zooepidemicus CR003, an in-depth analysis of hasB from CR003 was investigated in this work. HasB contains 401 amino acids with a molecular weight of 44.96 kDa, which exhibited 62.69 % homology with that from S. pyogenes. The optimal reaction condition for the purified hasB by heterologous expression was 30 °C and pH 9.0. Cu2+ and Zn2+ significantly inhibited hasB activity, whereas Mg2+ had a stimulatory effect. K m values of hasB for UDP-Glc and NAD+ were 0.1378 and 0.7583 mM, and V max were 391.3 and 548.8 μmol/min/mg, respectively. To our knowledge, it is currently the highest reported enzyme catalytic activity of hasB. Molecular docking showed that the binding energies of hasB with UDP-Glc and NAD+ were −731.01 and −314.17 kcal/mol, respectively, and the interaction forces between hasB and the substrate play a crucial role in stabilizing the conformation of protein-substrate complexes. Conclusively, hasB from CR003 showed high catalytic efficiency, which has great application potential for HA production. [Display omitted] • An in-depth analysis of hasB from S. zooepidemicus CR003 was investigated. • K m and V max values of hasB for UDP-Glc were 0.1378 mM and 391.3 μmol/min/mg, respectively. • Cu2+ and Zn2+ significantly inhibited hasB activity, whereas Mg2+ had a stimulatory effect. • It is currently the highest reported enzyme catalytic activity of hasB from S. zooepidemicus. [ABSTRACT FROM AUTHOR]

【Objective】This work is aimed at establishing a library of endogenous expression elements of Streptococcus epizooticus, and investigating the application of it in increasing hyaluronic acid yields.【Method】The 32 kinds of high, medium and low strength promoter and RBS combination (expression element, PR) were preliminarily screened, through transcriptomics analysis of S. zooepidemicus in the exponential or stationary stage of fermentation. Then, the strength of the 32 PRs was verified via detecting the transcriptional level of gfp gene and fluorescence intensity of GFP. Eventually, the key hyaluronic acid synthesis gene hasA, hasB, hasC, hasD, and hasE was overexpressed respectively with the selected strong expression element PR31, and the impact of the strong PR on enhancing hyaluronic acid production was assessed through a 2 L fermentation.【Result】The relative transcription of the aforementioned genes were elevated to 8.17, 7.32, 3.72, 39.48 and 9-fold. Among them, the overexpression of hasA and hasD increased the hyaluronic acid yield by 43% and 31%, respectively, compared to the wild type, reaching 5.654 g/L and 5.283 g/L. 【Conclusion】A library of endogenous expression elements of S. zooepidemicus had been constructed successfully, which can be employed for metabolic engineering, such as reinforcing the hyaluronic acid synthesis pathway or weaking competitive pathways. [ABSTRACT FROM AUTHOR]

Accurate and reliable estimation of specific growth rate (μ) in real-time is pivotal for reliable process monitoring of a bioprocess and subsequent implementation of advanced control strategies. Gibbs free energy dissipation is imminent for any biological system, and the metabolic heat flow measurements (calorimetry) formed the basis for estimating μ. However, the rationale behind selecting a suitable μ estimator model based on calorimetric perspective remains unexplored. The present investigation addresses the notion behind the selection of an appropriate estimator for μ and the assessment of the estimator models was illustrated using different types of energy metabolism, namely, high exothermic and low exothermic processes. The results indicated that the μ values from the instantaneous heat flow estimator significantly deviated (10-fold higher) from the offline values for highly exothermic process. Notably, the cumulative heat-based estimator accurately estimated μ values on both types of energy metabolism with performance metrics <0.005 h−1. [Display omitted] [ABSTRACT FROM AUTHOR]

Pyometra is an uncommon condition in mares and causes persistent (chronic) a accumulation of purulent material within the lumen of the uterus that lesd to grossly enlarged uterus. The possibility of recurrence after treatment is high. Here, we report pyometra in a 16-year-old Hanoverian mare with a history of infertility in the last 3 years despite repeated breeding, as well as a 15-year-old Thoroughbred mare with a 5 year history of infertility. Palpation of the uterus per rectum of the both mares revealed uniformly enlarged uterus located farther ventral in the abdomen. Ultrasonography revealed the uterus filled with echogenic fluid and no fetus could be seen. A corpus luteum (CI) was present on the left ovary. In the both mares, 3 days after prostaglandin injection transluminal adhesions of the cervix was broken down by digital palpation and 14 liter of milky creamy exudate from the uterus of Hanoverian mare and 8 liter from the uterus of Thoroughbred mare were collected using uterine flushing tube and then uterine lavage was performed. Bacteria isolation and identification in the both mares showed growth of Streptococcus equi subspecies zooepidemicus. Systemic antibiotic therapy, topical antimicrobial ointments for prevention of cervical adhesion and uterine lavage were performed frequently. [ABSTRACT FROM AUTHOR]

Mango processing generates significant amounts of residues (35–65%) that may represent environmental problems owed to improper disposal. The use of mango byproducts as substrates to produce hyaluronic acid (HA) is an attractive alternative to reduce the cost of substrate. In this study, we evaluated the potential of hydrolyzates from mango peels and seeds to produce HA by Streptococcus equi. subsp. zooepidemicus. The physicochemical characterization of mango residues showed that the seeds contain a higher amount of holocellulose (cellulose and hemicellulose), which amounts 54.2% (w/w) whereas it only represents 15.5% (w/w) in the peels. Mango peels, however, are composed mainly of hot water-extractives (62% w/w, that include sucrose, fructose, glucose and organic acids). A higher concentration of monosaccharides (39.8 g/L) was obtained from the enzymatic hydrolysis (with Macerex) of peels as compared to seeds (24.8 g/L with Celuzyme). From mango peels, hydrolyzates were obtained 0.6 g/L HA, while 0.9 g/L HA were obtained with hydrolyzates from mango seeds. These results demonstrate that mango byproducts have the potential to be used for production of HA. [ABSTRACT FROM AUTHOR]

In this research, we examined the production of hyaluronic acid (HA) by Streptococcus zooepidemicus strain MW26985 using different substrates and potato peel waste (PPW) as an affordable substrate. First, culture medium components, including carbon and nitrogen sources, were optimized for bacterial HA production. Five different carbon sources (glucose, sucrose, lactose, sago starch, and potato starch, at a concentration of 30 g/L) and three distinct nitrogen sources (peptone, yeast extract, and ammonium sulfate, at a concentration of 10 g/L) were investigated. Glucose, among the carbon sources, and yeast extract, among nitrogen sources, produced the most HA which was determined as 1.41 g/L. Afterward, potato peel sugars were extracted by dilute acid and enzymatic hydrolysis and then employed as a cost-effective carbon source for the growth of S. zooepidemicus. Based on the results, the fermentation process yielded 0.59 g/L HA from potato peel sugars through acid hydrolysis and 0.92 g/L HA from those released by enzymatic hydrolysis. The supplementation of both hydrolyzates with glucose as an additional carbon source enhanced HA production to 0.95 g/L and 1.18 g/L using acidic and enzymatic hydrolyzates, respectively. The cetyltrimethylammonium bromide (CTAB) turbidimetric method was used to evaluate the concentration of HA in the fermentation broth using the colorimetric method. Also, the peaks observed by Fourier transform infrared (FTIR) spectroscopy confirmed that the exopolysaccharide (EPS) was composed of HA. These observations demonstrate that potato peel residues can be a novel alternative as a carbon source for the economical production of HA by S. zooepidemicus. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Streptococcus equi subsp. zooepidemicus is a rare etiologic agent of bacterial meningitis in humans. The disease is a zoonotic infection and is transmitted through close contact with domestic animals, mainly horses. Only 37 cases of Streptococcus zooepidemicus meningitis have been reported in the literature until July 2023. The aim of this study is to present a rare clinical case of S. zooepidemicus-related meningitis in a human immunodeficiency virus (HIV)-positive patient and analyze the literature. We present a 23-year-old horse breeder patient with advanced immunosuppression due to acquired immunodeficiency syndrome (AIDS) and S. zooepidemicus meningitis, admitted at the Clinic of Infectious Diseases, St. George University Hospital, Plovdiv. The course of meningitis was severe since the beginning, with significant cerebral edema, disturbances in consciousness, persistent fever, and the development of complications against the background of AIDS-related conditions. S. zooepidemicus was microbiologically detected from cerebrospinal fluid culture. After prolonged treatment and a long hospital stay, the patient’s condition improved, and eventually he was discharged and recovered from the acute neuroinfection. Although extremely rare, S. zooepidemicus should be considered in patients with clinical and laboratory evidence of bacterial meningitis who have contact with animals, especially horses, other domestic animals, and their dairy products, as well as in immunocompromised patients. To the best of our knowledge, the current clinical case is the first report of S. zooepidemicus-related meningitis in a patient with HIV/AIDS.

This study describes an outbreak of Streptococcus equi subspecies zooepidemicus infections that caused meningoencephalitis and bacteremia related to unpasteurized milk consumption in northeastern Brazil. Epidemiological investigations and a brief literature review were conducted. Strains with possible neurotropism had not been identified in Brazil before these cases; however, in 2023, another case of meningoencephalitis caused by Streptococcus equi sp. zooepidemicus was described, revealing the need to maintain surveillance and highlighting that these neurotropic strains continue to circulate in the environment. [ABSTRACT FROM AUTHOR]

The manufacture, purification, and applications of hyaluronic acid (HA) are discussed in this article. Concerning the growing need for affordable, high-quality HA, it is essential to consider diverse production techniques using renewable resources that pose little risk of cross-contamination. Many microorganisms can now be used to produce HA without limiting the availability of raw materials and in an environmentally friendly manner. The production of HA has been associated with Streptococci A and C, explicitly S. zooepidemicus and S. equi. Different fermentation techniques, including the continuous, batch, fed-batch, and repeated batch culture, have been explored to increase the formation of HA, particularly from S. zooepidemicus. The topic of current interest also involves a complex broth rich in metabolites and residual substrates, intensifying downstream processes to achieve high recovery rates and purity. Although there are already established methods for commercial HA production, the anticipated growth in trade and the diversification of application opportunities necessitate the development of new procedures to produce HA with escalated productivity, specified molecular weights, and purity. In this report, we have enacted the advancement of HA technical research by analyzing bacterial biomanufacturing elements, upstream and downstream methodologies, and commercial-scale HA scenarios. [ABSTRACT FROM AUTHOR]

Streptococcus equi subsp. zooepidemicus is a rare etiologic agent of bacterial meningitis in humans. The disease is a zoonotic infection and is transmitted through close contact with domestic animals, mainly horses. Only 37 cases of Streptococcus zooepidemicus meningitis have been reported in the literature until July 2023. The aim of this study is to present a rare clinical case of S. zooepidemicus-related meningitis in a human immunodeficiency virus (HIV)-positive patient and analyze the literature. We present a 23-year-old horse breeder patient with advanced immunosuppression due to acquired immunodeficiency syndrome (AIDS) and S. zooepidemicus meningitis, admitted at the Clinic of Infectious Diseases, St. George University Hospital, Plovdiv. The course of meningitis was severe since the beginning, with significant cerebral edema, disturbances in consciousness, persistent fever, and the development of complications against the background of AIDS-related conditions. S. zooepidemicus was microbiologically detected from cerebrospinal fluid culture. After prolonged treatment and a long hospital stay, the patient's condition improved, and eventually he was discharged and recovered from the acute neuroinfection. Although extremely rare, S. zooepidemicus should be considered in patients with clinical and laboratory evidence of bacterial meningitis who have contact with animals, especially horses, other domestic animals, and their dairy products, as well as in immunocompromised patients. To the best of our knowledge, the current clinical case is the first report of S. zooepidemicus-related meningitis in a patient with HIV/AIDS. [ABSTRACT FROM AUTHOR]

Hyaluronic acid (HA), an anionic, non-sulfated glycosaminoglycan, has several clinical applications. This study examines several downstream methods for purifying HA with maximum recovery and purity. Following the fermentation of Streptococcus zooepidemicus MTCC 3523 to produce HA, the broth was thoroughly purified to separate cell debris and insoluble impurities using a filtration procedure and a variety of adsorbents for soluble impurities. Nucleic acids, proteins with high molecular weight, were successfully removed from the broth using activated carbons and XAD-7 resins. In contrast, insoluble and low molecular weight impurities were removed using diafiltration, with HA recovery of 79.16% and purity close to 90%. Different analytical and characterization procedures such as Fourier transform-infrared spectroscopy, X-ray diffraction, nuclear magnetic resonance, and scanning electron microscopy validated the presence, purity, and structure of HA. Microbial HA showed activity in tests for 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical-scavenging (4.87 ± 0.45 kmol TE/g), total antioxidant capacity (13.32 ± 0.52%), hydroxyl radical-scavenging (32.03 ± 0.12%), and reducing power (24.85 ± 0.45%). The outcomes showed that the precipitation, adsorption, and diafiltration processes are suitable for extracting HA from a fermented broth under the chosen operating conditions. The HA produced was of pharmaceutical grade for non-injectable applications. [Display omitted] • A statistical approach was modeled to improve the product yield, and different characterizations were performed. • Combined downstream processes improved the overall yield and purity of the final product. • Low energy usage throughout the purifying process was made possible by process intensification. • The purified hyaluronic acid showed antioxidant properties and was of pharmaceutical grade for non-injectable applications. [ABSTRACT FROM AUTHOR]

High broth viscosity due to the accumulation of hyaluronic acid (HA) causes a limited yield of HA. It is a major problem of HA production using Streptococcus zooepidemicus. Extractive fermentation via in situ product recovery (ISPR) was utilized to enhance the HA production. Resins from Amberlite: IRA400 Cl; IRA900 Cl; IRA410 Cl; IRA402 Cl; and IRA67 were tested for the HA adsorption. IRA67 showed high adsorption capacity on HA. The study of the adsorption via a 2 L stirred tank bioreactor of S. zooepidemicus fermentation was investigated to elucidate the adsorption of HA onto IRA67 in dispersed and integrated internal column systems. The application of a dispersed IRA67 improved the HA production compared to the fermentation without resin addition by 1.37-fold. The HA production was further improved by 1.36-fold with an internal column (3.928 g/L) over that obtained with dispersed IRA67. The cultivation with an internal column shows the highest reduction of viscosity value after the addition of IRA67 resin: from 58.8 to 23.7 (mPa·s), suggesting the most effective ISPR of HA. The improved biosynthesis of HA indicated that an extractive fermentation by ISPR adsorption is effective and may streamline the HA purification. [ABSTRACT FROM AUTHOR]

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Hyaluronic acid is a polysaccharide endowed with distinctive biological and physiological competencies. Given its queer properties, hyaluronic acid has exclusive praxis in the cosmetics and medical industries. The surmounting demand for hyaluronic acid is the propulsion behind the necessity for finding the amenable ways for its production. Fermentation progression of Streptococcus zooepidemicus is reckoned as the superlative prompt and ambient approach for hyaluronic acid fabrication. For the unabated advancements in the industrial production of hyaluronan, industrial byproducts utilization is a fateful stile. The recent perusal is to optimize the fermentation production conditions of hyaluronic acid using cane molasses (a byproduct of sugar production) as a carbon source. The impact of different ranges of temperatures (33–41 °C), pH (6–8), and agitation rates (100–250 rpm) on the production process was calibrated using RSM using CCD as a statistical modality. In a 3.7 L bioreactor, 3.31 g/L hyaluronic acid was achieved at 9.74 percent molasses, 36.2 °C, pH 6.46, and a 207 rpm agitation rate using a batch fermentation technique. With a pH of 7, HPLC was conducted at 25 °C using a C18 column at a rate of 0.8 ml/min, and the wavelength was determined using a UV detector. The average retention time was 2.202 min. The FT-IR spectrum's output was also observed, and it matched the standard hyaluronic acid well. [ABSTRACT FROM AUTHOR]

Streptococcus zooepidemicus, group C Streptococci, is currently used for the industrial production of hyaluronic acid (HA). However, genetic manipulation of S. zooepidemicus is severely limited by its low transformation efficiency, which might be in part due to the Restriction–Modification (R–M) systems. The complete genome sequence of S. zooepidemicus ATCC39920 revealed the presence of two putative R–M systems, type I and type II. The putative type I R–M system is encoded by three closely linked genes: hsdR (SeseC_01315), hsdS, hsdM (SeseC_01318), and the putative type II R–M system consists of two closely linked genes: SeseC_02360 and yhdJ (SeseC_02362). Inactivation of hsdR, encoding the restriction endonuclease (REase) of the type I R–M system, showed no apparent effects on transformation efficiency, implying that disarmament of the type I R–M system alone is not sufficient for increasing transformation efficiency. However, inactivation of SeseC_02360, encoding the REase of the type II R–M system, improved transformation efficiency by 4.97 folds, indicating that type II R–M system is the major barrier that restricts genetic transformation in S. zooepidemicus. Furthermore, S. zooepidemicus strains lacking either of the two R–M systems are phenotypically indistinguishable from the wild-type in terms of cell growth and HA production. In summary, our study revealed that the type II R–M system is the main barrier to genetic transformation in S. zooepidemicus ATCC39920, and that the deletion of the type II R–M system renders S. zooepidemicus more transformable, thus facilitating metabolic engineering of this industrially important microorganism. [ABSTRACT FROM AUTHOR]

Streptococcus equi subsp. zooepidemicus (SEZ) is a highly contagious infectious organism that causes disease in horses. SEZ is seldom isolated from humans; however, zoonotic infections are occasionally reported in individuals exposed to horses and other livestock. Herein, we report three human cases of SEZ in individuals, one with direct horse contact and two among individuals who had eaten raw horse meat. The phylogenetic tree showed that the genotypes of SEZ isolates from two of the cases on Jeju Island, South Korea, were similar to those of isolates from the United Kingdom and the United States of America. [ABSTRACT FROM AUTHOR]

Bored coffee beans (BCBs) are the residues left from the pest Hypothenemus hampei that attacks coffee crops, resulting in enormous economic losses. The bioconversion of monosaccharides from BCBs into hyaluronic acid (HA) is appealing both for using the residues and given the high commercial value of HA. This study dealt with the production of HA using Streptococcus zooepidemicus by employing either acid (AcH) or enzymatic (EnH) hydrolyzates from BCBs. The highest release of monosaccharides (evaluated using surface response methodology) was obtained with EnH (36.4 g/L); however, S. zooepidemicus produced more HA (1.5 g/L) using AcH compared to EnH. Hydrolyzates from acetone-extracted BCBs yielded 2.7 g/L of HA, which is similar to the amount obtained using a synthetic medium (2.8 g/L). This report demonstrates the potential of hydrolyzates from bored coffee beans to produce HA by S. zooepidemicus. [ABSTRACT FROM AUTHOR]

Peptones are widely used as complex fermentation substrate for hyaluronic acid (HA) production; however, use of peptones in HA production reduces commercial competitiveness due to their high price. The present study was conducted to test the feasibility of sheep wool peptone (SWP) (mainly organic nitrogen source) and molasses (mainly carbon source) as cheap substrates for HA production from Streptococcus zooepidemicus ATCC 35246. Six peptones (SWP I–VI) were prepared from sheep wool using different chemical hydrolysis methods. Among them, SWP-VI was determined to be more fovarable for HA production. SWP-VI was compared with commercial tryptone peptone (TP) and protease peptone (PP) in order to evaluate its effectiveness in production of HA, lactic acid (LA) and cell biomass (CB). The protein contents of SWP-VI, TP and PP were determined as 70.6, 83.1 and 83.8 g/100 g, respectively. The best peptone for HA and CB production was SWP-VI, whereas PP was found to be more favourable for LA production. Maximum HA concentrations in SWP-VI, TP and PP media were determined as 3.54, 2.58 and 2.47 g/L, respectively. [ABSTRACT FROM AUTHOR]

Hyaluronic acid (HA) is a molecule of significant commercial value with applications in the pharmaceutical, cosmetic, and medical industries. It is typically produced through microbial fermentation. However, the efficiency of this type of process can be affected by inhibition phenomena due to the substrate or the product. In this sense, the effect of different initial concentrations of glucose (9.3–59.9 g/L) and lactic acid (10.7–56.0 g/L) was studied in the present investigation to determine their effect on the growth of Streptococcus zooepidemicus and HA production in batch fermentation. The maximum HA concentration obtained was 3.3 g/L from 59.9 g/L glucose, while productivity, yield, and specific rate of HA formation were higher at 31.9 g/L glucose. The biomass produced followed a stepwise pattern with increasing glucose, although the specific growth rate decreased after reaching 39.1 g/L glucose. The initial presence of lactic acid (LA) hurt fermentation, decreasing the amount of biomass and HA produced; the inhibitory effect of LA was more pronounced at 19.4 g/L and above, decreasing productivities, yields, and specific rates. Compared to experimental data, the proposed mathematical model described satisfactorily (R 2 > 0.9) the dynamics of the fermentation process at different culture conditions. [Display omitted] • The initial concentration of glucose reduces the specific growth rate above 31.9 g/L • Growth and hyaluronic acid production were strongly inhibited by the initial addition of lactic acid • Inhibition by the initial lactic acid did not affect the production of the acid itself • The kinetic model developed adequately predicts the culture dynamics [ABSTRACT FROM AUTHOR]

Streptococcus equi. subsp. zooepidemicus (S. zooepidemicus) associated diseases in dogs have emerged as a significant concern over recent decades. S. zooepidemicus occurs sporadically in dog populations globally, with increased prevalence in shelters/kennels. This study used multilocus sequence typing (MLST) of 149 independent canine S. zooepidemicus isolates to assess associations between sequence type and breed, country of origin, disease severity, sampling type, year, and behaviour within an outbreak. No clear associations for breed, country, sampling type and year were determined in this study. ST-10 and 123 strains were present within all disease categories, from no clinical signs to severe disease. Assessment of S. zooepidemicus infection in 3 UK outbreaks at the same location found ST-10, 18, 123 strains, and a ST-173 strain in a US outbreak, were associated with haemorrhagic pneumonia and persisted in kennelled populations over time. The ST-173 clonal complex has been noted to have severe virulence capabilities in dogs and other species. S. zooepidemicus seems to thrive in environments with a high risk of transmissibility, overcrowding, stress and naïve populations, particularly for those in shelters/kennels. MLST alone cannot determine the virulence phenotype of S. zooepidemicus in dogs. However, a level of conservancy and diversity within ST allelic loci aids the opportunity to cause severe disease in dogs. Thus, further research into whole genome sequencing and characterising the virulence factors of S. zooepidemicus is warranted in dogs. • Streptococcus zooepidemicus has been isolated in outbreaks of canine pneumonia. • MLST allelic loci have been associated with varying clinical severity of disease. • ST10, ST18, ST123 and ST173 have been linked to outbreaks in kennelled dogs • MLST alone cannot determine the virulence of streptococcus zooepidemicus. [ABSTRACT FROM AUTHOR]

The hyaluronic acid (HA) global market growth can be attributed to its use in medical, cosmetic, and pharmaceutical applications; thus, it is important to have validated, analytical methods to ensure confidence and security of its use (and to save time and resources). In this work, a size-exclusion chromatography method (HPLC-SEC) was validated to determine the concentration and molecular distribution of HA simultaneously. Analytical curves were developed for concentration and molecular weight in the ranges of 100–1000 mg/L and 0.011–2.200 MDa, respectively. The HPLC-SEC method showed repeatability and reproducibility greater than 98% and limits of detection and quantification of 12 and 42 mg/L, respectively, and was successfully applied to the analysis of HA from a bacterial culture, as well as cosmetic, and pharmaceutical products.

ABSTRACT Streptococcus equi subspecies zooepidemicus (SEZ) are group C streptococci that are important pathogens of economically valuable animals such as horses and pigs. Here, we found that many SEZ isolates bind to a monoclonal antibody that recognizes poly-N-acetylglucosamine (PNAG), a polymer that is found as a surface capsule-like structure on diverse microbes. A fluorescence-activated cell sorting-based transposon insertion sequencing (Tn-seq) screen, coupled with whole-genome sequencing, was used to search for genes for PNAG biosynthesis. Surprisingly, mutations in a gene encoding an M-like protein, szM, and the adjacent transcription factor, designated sezV, rendered strains PNAG negative. SezV was required for szM expression and transcriptome analysis showed that SezV has a small regulon. SEZ strains with inactivating mutations in either sezV or szM were highly attenuated in a mouse model of infection. Comparative genomic analyses revealed that linked sezV and szM homologues are present in all SEZ, S. equi subspecies equi (SEE), and M18 group A streptococcal (GAS) genomes in the database, but not in other streptococci. The antibody to PNAG bound to a wide range of SEZ, SEE, and M18 GAS strains. Immunochemical studies suggest that the SzM protein may be decorated with a PNAG-like oligosaccharide although an intact oligosaccharide substituent could not be isolated. Collectively, our findings suggest that the szM and sezV loci define a subtype of virulent streptococci and that an antibody to PNAG may have therapeutic applications in animal and human diseases caused by streptococci bearing SzM-like proteins. IMPORTANCE M proteins are surface-anchored virulence factors in group A streptococci, human pathogens. Here, we identified an M-like protein, SzM, and its positive regulator, SezV, in Streptococcus equi subspecies zooepidemicus (SEZ), an important group of pathogens for domesticated animals, including horses and pigs. SzM and SezV homologues were found in the genomes of all SEZ and S. equi subspecies equi and M18 group A streptococcal strains analyzed but not in other streptococci. Mutant SEZ strains lacking either sezV or szM were highly attenuated in a mouse model of infection. Collectively, our findings suggest that SezV-related regulators and the linked SzM family of M-like proteins define a new subset of virulent streptococci.

Streptococcus zooepidemicus is an important opportunistic pathogen of several species including humans. This organism is also well-known as the main producing strain in industrial production of hyaluronic acid (HA), which is the component of its capsule polysaccharide. How its virulence and capsule polysaccharide production is regulated remains poorly understood. Intercellular chemical signaling among bacteria provides communities of microbes the opportunity to coordinate gene expression to facilitate group behavior, such as pathogenicity, capsule polysaccharide production, etc. Yet no conserved cell-to-cell signaling system has been elucidated in S. zooepidemicus. Encoded within the genome of S. zooepidemicus is one Rgg regulator encoding gene (rgg) with low similarity to both rgg2 and rgg3 from Streptococcus pyogenes. A small ORF (named as shp) encoding a novel short hydrophobic peptide (SHP) was found in the vicinity of rgg. We found that the active form of pheromone is short and hydrophobic (LLLLKLA), corresponding to the C terminal 7 amino acids of the pre-peptide Shp, which shows divergent sequence to all peptide pheromones reported in streptococci. In response to active SHP, Rgg functions as a transcriptional activator to induce the expression of shp, forming a positive feedback circuit. Bacteria social behaviors, such as capsule polysaccharide production and biofilm formation, were significantly affected when the rgg-shp pathway was inactivated. These data provide the first demonstration that Rgg/Shp signaling pathway comprises an active quorum sensing system in S. zooepidemicus.

Streptococcus equi subspecies zooepidemicus ( S. zooepidemicus ) is a zoonotic pathogen that primarily infects horses, pigs, and dogs. Although rare, it has also been shown to infect humans who consume unpasteurized dairy food or have direct contact with horses. Here, we present a case of S. zooepidemicus bacteremia in a patient without a clear mode of transmission. An 86-year-old male with a past medical history of coronary artery disease, heart failure with reduced ejection fraction, complete heart block status post pacemaker, hypertension, hyperlipidemia, and type 2 diabetes mellitus presented to the Emergency Department with fever and chills. He had fevers and rigors for three days but denied weight loss, cough, sore throat, or rashes. In the Emergency Department, vital signs revealed a fever of 101.2 degrees Fahrenheit and a heart rate of 110 with other stable vital signs. The physical exam was unremarkable except for tachycardia, and laboratory work revealed no leukocytosis but elevated inflammatory markers and elevated lactate. Computed tomography of the chest, abdomen, and pelvis did not reveal any source of infection. Blood cultures grew S. zooepidemicus and the Infectious Diseases team was consulted, who started the patient on Penicillin G. Due to concern for pacer-lead infective endocarditis, transthoracic and transesophageal echocardiograms were performed, which did not show valvular vegetations. Repeat blood cultures showed clearance of the infection, and the patient was ultimately discharged on amoxicillin. While our patient denied consuming unpasteurized dairy products or having direct contact with horses, upon further questioning, he did endorse family members who occasionally interacted with horses. This case is valuable as it adds to the sparse literature on S. zooepidemicus infections specifically in humans. Extensive history taking is of utmost importance when a clear source of infection is not easily identifiable. Further research is also needed to better understand the various modes of transmission of this bacterium to better target and caution those at an increased risk of infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Shah et al.)

Bored coffee beans (BCBs) are the residues left from the pest Hypothenemus hampei that attacks coffee crops, resulting in enormous economic losses. The bioconversion of monosaccharides from BCBs into hyaluronic acid (HA) is appealing both for using the residues and given the high commercial value of HA. This study dealt with the production of HA using Streptococcus zooepidemicus by employing either acid (AcH) or enzymatic (EnH) hydrolyzates from BCBs. The highest release of monosaccharides (evaluated using surface response methodology) was obtained with EnH (36.4 g/L); however, S. zooepidemicus produced more HA (1.5 g/L) using AcH compared to EnH. Hydrolyzates from acetone-extracted BCBs yielded 2.7 g/L of HA, which is similar to the amount obtained using a synthetic medium (2.8 g/L). This report demonstrates the potential of hydrolyzates from bored coffee beans to produce HA by S. zooepidemicus.

Polysaccharide peptides (or protein-bound polysaccharides, PSPs) are commonly found in mushrooms and plants and possess important nutritional properties and health benefits. The pathogenic bacterium Streptococcus zooepidemicus does not inherently produce PSPs but secretes the capsular polysaccharide hyaluronan. However, in a previous investigation of the catalytic mechanism of UDP-glucose dehydrogenase (UGDH), a PSP of peptide-bound hyaluronan was found to be produced by S. zooepidemicus through the in vivo expression of a mutant of the gene encoding UGDH. In the present study, this hyaluronan-derived PSP was structurally characterized by FT-IR, NMR, and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the data confirmed that the polysaccharide backbone, hyaluronan, is covalently bound to the side-chain peptides via an amide linkage. More importantly, the bacterial production of a PSP via this genetic modification method should inspire further research on the in vitro enzymatic synthesis of PSPs or even naturally occurring polysaccharide derivatives and may provide a theoretical foundation for investigating the in vivo synthetic mechanism of PSPs. Image 1 • Streptococcus zooepidemicus inherently secretes the polysaccharide hyaluronan. • A mutant strain of S. zooepidemicus produces a polysaccharide peptide (PSP). • This artificial PSP is verified to be a hyaluronan-based derivative. • Production of PSP via this genetic modification is novel and illuminating. [ABSTRACT FROM AUTHOR]

Streptococcus zooepidemicus is an important opportunistic pathogen of several species including humans. This organism is also well-known as the main producing strain in industrial production of hyaluronic acid (HA), which is the component of its capsule polysaccharide. How its virulence and capsule polysaccharide production is regulated remains poorly understood. Intercellular chemical signaling among bacteria provides communities of microbes the opportunity to coordinate gene expression to facilitate group behavior, such as pathogenicity, capsule polysaccharide production, etc. Yet no conserved cell-to-cell signaling system has been elucidated in S. zooepidemicus. Encoded within the genome of S. zooepidemicus is one Rgg regulator encoding gene (rgg) with low similarity to both rgg2 and rgg3 from Streptococcus pyogenes. A small ORF (named as shp) encoding a novel short hydrophobic peptide (SHP) was found in the vicinity of rgg. We found that the active form of pheromone is short and hydrophobic (LLLLKLA), corresponding to the C terminal 7 amino acids of the pre-peptide Shp, which shows divergent sequence to all peptide pheromones reported in streptococci. In response to active SHP, Rgg functions as a transcriptional activator to induce the expression of shp , forming a positive feedback circuit. Bacteria social behaviors, such as capsule polysaccharide production and biofilm formation, were significantly affected when the rgg - shp pathway was inactivated. These data provide the first demonstration that Rgg/Shp signaling pathway comprises an active quorum sensing system in S. zooepidemicus. [ABSTRACT FROM AUTHOR]

This work investigates the production of hyaluronic acid (H) by Streptococcus equi subsp. zooepidemicus in complex media formulated with peptones obtained from Scyliorhinus canicula viscera by-products. Initially, in batch cultures, the greatest productions were achieved using commercial media (3.03 g/L) followed by peptones from alcalase hydrolyzed viscera (2.32 g/L) and peptones from non-hydrolyzed viscera (2.26 g/L). An increase of between 12% and 15% was found in subsequent fed-batch cultures performed on waste peptones. Such organic nitrogen sources were shown to be an excellent low-cost substrate for microbial H, saving more than 50% of the nutrient costs.

Abstract A 2-month-old Andalusian colt in central Spain presented dyspnea, diarrhea, and depression. Despite the initial treatment for presumptive pneumonia caused by Rhodococcus equi, after 1 hour, the colt showed signs of shock and died. The necropsy revealed multiple abscesses in the lung, as well as enlarged mesenteric lymph nodes, and hemorrhagic peritonitis. Samples were taken from trachea, lung parenchyma, small intestine, and colon. All samples yielded a pure culture of Streptococcus equi spp. zooepidemicus, whereas R. equi was excluded by using a specific qPCR performed on all samples. Isolates from all samples were characterized by pulse-field gel electrophoresis and the representative strain was identified by MALDI-TOF and qPCR. Its genome was sequenced, assembled, and annotated; multilocus sequence typing data assigned the strain to sequence type 70 and genes related to adhesion, bacteriocins, invasion, antimicrobial resistance, toxins, and superantigens szeN and szeP were identified in the assembled genome. The strain was susceptible to all antimicrobials tested. Our findings support the inclusion of S. zooepidemicus on an endemic farm where R. equi has been ruled out via PCR testing. S. zooepidemicus can cause multiorgan disease in foals, similar to infection in humans. The presence of superantigens in this S. zooepidemicus strain may have contributed to the fatal outcome in this clinical case. Highlights • Streptococcus zooepidemicus should be in the differential diagnoses when R. equi is ruled out by PCR testing. • S. zooepidemicus can cause multiorgan disease in foals, similar to infection in humans. • The presence of superantigens in the strain may have contributed to fatal outcome. [ABSTRACT FROM AUTHOR]

In this study, we evaluated palmyra palm sugar (PJ) formulated medium for HA production using Streptococcus zooepidemicus MTCC 3523. Concurrently, effect of various carbon and nitrogen sources, an analysis of the effect of initial sugar concentration, on HA production were examined. The PJ - soya peptone based medium was found to be the most effective, and resulted in 0.54 ± 0.08 g L -1 of HA titer and specific growth rate of 0.5 ± 0.07 h -1 in shake-flask experiments. Kinetic experiments were performed in a bioreactor, and the initial PJ concentrations were varied from 10 - 50 g L -1 . Maximum HA and LA titers (1.22 g L -1 and 20. 2 g L -1 ) were obtained at 30 g L -1 PJ concentration in bioreactor. Han–Levenspiel substrate inhibition model exhibited best fit for the experimental data. The growth inhibitory PJ concentration was found to be at 90.5 g L -1 . Medium based process economic analysis proved PJ and soya peptone based medium as most economical for the production of HA. The present investigation validates the potential of PJ and soya peptone based medium for enhanced economical production of HA. This study will also provide new product dimensions and market avenues, to the marginalized palmyra palm farmers and toddy tapping community, who are unable to secure the right price for their product. [ABSTRACT FROM AUTHOR]

ABSTRACT Iceland is free of the major infectious diseases of horses. However, in 2010 an epidemic of respiratory disease of unknown cause spread through the country’s native horse population of 77,000. Microbiological investigations ruled out known viral agents but identified the opportunistic pathogen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) in diseased animals. We sequenced the genomes of 257 isolates of S. zooepidemicus to differentiate epidemic from endemic strains. We found that although multiple endemic clones of S. zooepidemicus were present, one particular clone, sequence type 209 (ST209), was likely to have been responsible for the epidemic. Concurrent with the epidemic, ST209 was also recovered from a human case of septicemia, highlighting the pathogenic potential of this strain. Epidemiological investigation revealed that the incursion of this strain into one training yard during February 2010 provided a nidus for the infection of multiple horses that then transmitted the strain to farms throughout Iceland. This study represents the first time that whole-genome sequencing has been used to investigate an epidemic on a national scale to identify the likely causative agent and the link to an associated zoonotic infection. Our data highlight the importance of national biosecurity to protect vulnerable populations of animals and also demonstrate the potential impact of S. zooepidemicus transmission to other animals, including humans. IMPORTANCE An epidemic of respiratory disease affected almost the entire native Icelandic horse population of 77,000 animals in 2010, resulting in a self-imposed ban on the export of horses and significant economic costs to associated industries. Although the speed of transmission suggested that a viral pathogen was responsible, only the presence of the opportunistic pathogen Streptococcus zooepidemicus was consistent with the observed clinical signs. We applied genomic sequencing to differentiate epidemic from endemic strains and to shed light on the rapid transmission of the epidemic strain throughout Iceland. We further highlight the ability of epidemic and endemic strains of S. zooepidemicus to infect other animals, including humans. This study represents the first time that whole-genome sequencing has been used to elucidate an outbreak on a national scale and identify the likely causative agent.

Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) causes outbreaks of fatal respiratory disease in dog shelters and fatal respiratory and neurologic disease in cat shelters. We conducted multi-locus sequence typing analysis on S. zooepidemicus isolates from 5 Canadian and 3 Israeli cats with severe respiratory and neurologic disease, plus 1 isolate from a clinically normal shelter cat. Our aim was to determine if feline outbreaks are clonal and whether there is commonality between feline and canine strains. ST363 was identified as the causative strain of a Canadian outbreak of S. zooepidemicus–linked disease, and is a double-locus variant of ST173, which was isolated from one of the Israeli cats. ST363 was also isolated from the clinically normal cat, indicative of the potential for enzootic infection in shelters. Strains within the ST173 clonal complex were responsible for 2 large canine outbreaks in the United States and the United Kingdom, as well as the death of 1 cat in the United States outbreak. ST215 was isolated from 2 cats in the Israeli outbreak, and is unrelated to the ST173 complex. We conclude that S. zooepidemicus outbreaks in cat shelters are clonal and that strains within the ST173 clonal complex are pathogenic for both dogs and cats. [ABSTRACT FROM AUTHOR]

Streptococcus zooepidemicus (Sz) is a tonsillar commensal of horses that opportunistically invades the respiratory tract of weanling foals causing purulent rhinitis and tracheobronchitis. Ten weanling foals in an isolated herd with a history of Sz-associated respiratory disease in recent years were immunized with recombinant proteins SzPNC78, endopeptidase, and hyaluronidase prior to weaning to determine the effect of serum antibodies on populations of nasopharyngeal Sz and mucopurulent nasal discharges. Levels of serum antibodies to all three proteins were significantly ( P ≤ .01) increased compared to levels in 10 nonimmunized control foals at weaning (day 0). Numbers of Sz in nasopharyngeal washes of control foals were initially higher on day 35 than in washes of immunized foals. However, this difference was not maintained, and counts in washes of both groups were similar over the following 7 weeks. By day 104 postweaning, Sz was no longer detected in washes of six foals in each group. Streptococcus zooepidemicus (ST-45) expressing a specific protective protein (SzP) phenotype dominated from days 35 to 104 in both groups. A second clone, ST-10, was detected intermittently in five immunized and two control foals. Mucopurulent nasal discharges of seven immunized and eight control foals on days 78 and 85 were associated with counts of Sz that ranged as high as 10 6 colony-forming units/mL and with significantly increased levels of SzP-specific IgG in washes. Increase in specific IgG but not IgA was temporary suggesting transudation through inflamed mucosa. This increase was generally greater in washes of immunized (mean enzyme-linked immunosorbent assay optical density [ELISA OD] = 1.73) than control (mean ELISA OD = 1.08) foals suggesting a benefit of systemic immunization may be therapeutic by enhancing clearance of Sz following inflammation provoked by local invasion. [ABSTRACT FROM AUTHOR]

The zoonotic bacterium Streptococcus equi subsp . zooepidemicus ( S. zooepidemicus ) is a diverse, opportunistic pathogen that can cause mastitis in dairy sheep and goats. We used multilocus sequence typing (MLST) to define the genetic diversity of 60 isolates of S. zooepidemicus , which were recovered from sheep and goats in Spain between 2003 and 2010. We identify a novel clonal complex based on sequence type (ST), ST-236, which accounted for 39 of the 60 isolates. A representative ST-236 strain, S. zooepidemicus strain C7 ( Sz C7), was sequenced and interrogated for the presence of novel nutritional uptake or utilisation systems, the acquisition of which have previously been shown to be important for environmental adaptation in other streptococcal pathogens. A novel phosphoenolpyruvate sugar phosphotransferase system (PTS), which enabled the utilisation of arbutin, was identified. Functionality of the PTS was confirmed following deletion of the PTS from Sz C7. Arbutin is found in multiple animal foodstuffs and we propose that the ability to utilise arbutin may have conferred a selective advantage to strains infecting animals, the diet of which contains this sugar. [ABSTRACT FROM AUTHOR]

Despite the significant potential of Streptococcus zooepidemicus for hyaluronic acid (HA) production with high molecular weight (MW), the HA degrading properties of hyaluronidase prevents the bacteria to achieve enhanced HA yield with high MW. In the present study, we aim to knockout the hyaluronidase enzyme and assess its effects on the yield and MW of the produced HA. The kanamycin resistance gene between the left and right arms of hyaluronidase gene was inserted into pUC18 plasmid to construct pUC18-L-kana-R as a recombinant suicide plasmid. The construct was then transferred into S. zooepidemicus to induce the homologous recombination between the hyaluronidase gene and the kanamycin resistance gene. Gene deletion was confirmed by PCR and enzyme assay. The product was cultured on selectable medium in which the MW of HA was increased from 1.5 to 3.8 MDa. The yield of HA production using the mutant strain was higher in all different concentrations of glucose from 40 to 120 g/l. Moreover, glucose increase results in higher HA production within both wild-type and recombinant strains. However, the growth rate of HA concentration (the slope of the plot), as a consequence of increased glucose concentration, is always higher for the recombinant strain. Unlike the wild-type strain, there was no sharp HA production drop approaching the 6 g/l HA concentration. In conclusion, hyaluronidase activity and HA concentration and MW exhibited a mutual control on each other. Based on our results, deletion of the hyaluronidase gene positively affects the yield and MW of HA. [ABSTRACT FROM AUTHOR]

This study reveals the genetic and biochemical changes underlying the enhanced hyaluronan (HA) biosynthesis in Streptococcus zooepidemicus. After multiple rounds of atmospheric and room temperature plasma (ARTP) mutagenesis combined with novel bovine serum albumin/cetyltrimethylammonium bromide coupled high-throughput screening assay, the HA yield of the mutant was increased by 42.9% and reached 0.813 g L−1 with a molecular weight of 0.54 × 106 Da within 18 h by shaking flask culture. HA production was increased to 4.56 g L−1 by batch culture in 5-L fermenter. Transcriptome sequencing exhibits that distinct mutants have similar genetic changes. Regulation in direction of metabolic flow into the HA biosynthesis, by enhancing genes responsible for the biosynthesis of HA including hasB , glmU and glmM , weaking downstream gene (nagA and nagB) of UDP-GlcNAc and significantly down-regulating transcription of wall-synthesizing genes, resulting in the accumulation of precursors (UDP-GlcA and UDP-GlcNAc) increased by 39.74% and 119.22%, respectively. These associated regulatory genes may provide control point for engineering of the efficient HA-producing cell factory. [Display omitted] • ARTP mutagenesis combining with novel coupled screening assay were performed. • After multiple rounds of mutagenesis, the HA yield was increased by 42.9 %. • Distinct mutants have similar genetic changes responded to HA accumulation trends. • UDP-GlcA and UDP-GlcNAc accumulation increased by 39.74 % and 119.22 %, respectively. • Relevant enzymes and genetic variation responsible for HA biosynthesis were analyzed. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two-stage semi-continuous fermentation was developed for hyaluronic acid production. • High hyaluronic acid titer and productivity can be attained in 2nd stage bioreactor. • Addition of hyaluronidase can reduce viscosity of broth and improve production. Hyaluronic acid is a kind of mucopolysaccharide that has wide applications in cosmetics, health food, and orthopedics. Using Streptococcus zooepidemicus ATCC 39920 as parent, a beneficial mutant SZ07 was obtained by UV mutagenesis, giving 1.42 g/L hyaluronic acid in shake flasks. To enhance the efficiency of hyaluronic acid production, a semi-continuous fermentation process consisted of two-stage 3–L bioreactors was developed, in which 1.01 g/L/h productivity and 14.60 g/L hyaluronic acid were obtained. To further enhance the titer of hyaluronic acid, recombinant hyaluronidase Sz HYal was added into 2nd stage bioreactor at 6 h to reduce the viscosity of broth. The highest hyaluronic acid titer of 29.38 g/L was achieved with a productivity of 1.13 g/L/h at 300 U/L Sz HYal after 24 h. This newly developed semi-continuous fermentation process provides a promising strategy for the industrial production of hyaluronic acid and related polysaccharides. [ABSTRACT FROM AUTHOR]

Streptococcus equi subspecies zooepidemicus is a pathogen of veterinary interest that causes disease in horses, pigs, and dogs and is recognized as an emerging cause of feline respiratory disease. Human zoonotic disease is rare but can occur in patients who are taking care of horses and via consumption of unpasteurized animal products. We describe a case of soft tissue infection and bacteremia in an elderly patient who had contact with a cat presenting respiratory symptoms and was treated with antibiotics. To the best of our knowledge, this is the first description of cat-to-human transmission of Streptococcus zooepidemicus ., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Dolapsakis et al.)