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.

SzM protein of Streptococcus equi ssp. zooepidemicus triggers the release of neutrophil extracellular traps depending on GSDMD.

Streptococcus equissp.zooepidemicus (SEZ) is a crucial pathogen and contributes to various infections in numerous animal species. Swine streptococcicosis outbreak caused by SEZ has been reported in several countries in recent years. SzM protein is a cell membrane-anchored protein, which exhibits as an important virulence factor of SEZ. Effects of SzM protein on host innate immune need further study. Here, recombinant SzM (rSzM) protein of the SEZ was obtained, and mice were intraperitoneally injected with rSzM protein. We discovered that rSzM protein can recruit neutrophils into the injected site. In further study, neutrophils were isolated and treated with rSzM protein, NETs release were triggered by rSzM protein independently, and GSDMD protein was promoted-expressed and activated. In order to investigate the role of GSDMD in NETs formation, neutrophils isolated from WT mice and GSDMD-/- mice were treated with rSzM protein. The results showed that GSDMD deficiency suppressed the NETs release. In conclusion, SzM protein of SEZ can trigger the NETs release in a GSDMD-depending manner.

Hyaluronic acid production by Streptococcus zooepidemicus MW26985 using potato peel waste hydrolyzate.

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.

Untangling the stranglehold through mathematical modelling of Streptococcus equi subspecies equi transmission.

Strangles, a disease caused by infection with Streptococccus equi subspecies equi (S. equi), is endemic worldwide and one of the most frequently diagnosed infectious diseases of horses. Recent work has improved our knowledge of key parameters of transmission dynamics, but important knowledge gaps remain. Our aim was to apply mathematical modelling of S. equi transmission dynamics to prioritise future research areas, and add precision to estimates of transmission parameters thereby improving understanding of S. equi epidemiology and quantifying the control effort required. A compartmental deterministic model was constructed. Parameter values were estimated from current literature wherever possible. We assessed the sensitivity of estimates for the basic reproduction number on the population scale to varying assumptions for the unknown or uncertain parameters of: (mean) duration of carriership (1∕γC), relative infectiousness of carriers (f), proportion of infections that result in carriership (p), and (mean) duration of immunity after natural infection (1∕γR). Available incidence and (sero-)prevalence data were compared to model outputs to improve point estimates and ranges for these currently unknown or uncertain transmission-related parameters. The required vaccination coverage of an ideal vaccine to prevent major outbreaks under a range of control scenarios was estimated, and compared available data on existing vaccines. The relative infectiousness of carriers (as compared to acutely ill horses) and the duration of carriership were identified as key knowledge gaps. Deterministic compartmental simulations, combined with seroprevalence data, suggest that 0.05

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

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

Invitro antibacterial activity of bark, leaf and root extracts of combretum molle plant against streptococcus equi isolated from clinical cases of strangles in donkeys and horses.

BACKGROUND: Effective therapy for many infections is becoming difficult due to the evolutionary development of drug resistance, and hence, the development of alternative treatment options mainly from herbs is crucial. The objective of this study was to investigate the antibacterial effects of ethanol extracts of stem bark, leaves and roots of Combretum molle against Streptococcus equi isolated from clinical cases of strangles using in vitro tests. METHODS: Plant extraction was performed using a maceration technique with 80% ethanol. The mean zone of inhibition was determined using the agar well diffusion method. Six serial dilutions with different concentrations (10%, 5%, 2.5%, 1.25%, 0.625% and 0.3125%) of each plant extract were prepared using dimethyl sulfoxide (DMSO). A modified agar microdilution method was used to determine the minimum inhibitory concentration (MICs) of the extracts. RESULTS: The results revealed that all plant extracts showed significant antibacterial activity. The root extract showed the best antibacterial effect compared to the others at all concentrations, with MZI values of 27.5, 23.225, 20.5, 17.9, 15.65 and 12.25 for the respective concentrations mentioned above and an MIC of 250 µg/ml. It was followed by the stem bark extract, which had MZI values of 24.67, 22.35, 18.225, 16.175, 11.125 and 8.2 millimeters and an MIC of 375 µg/ml. The leaf extract also had significant activity, with MZI values of 20.175, 18.25, 15.7, 13.125, 9.4 and 6.75 in millimeters and an MIC of 500 µg/ml. There was a direct relationship between the concentrations of the plant extracts and the level of inhibition. CONCLUSION: The test plant extracts were compared with the conventional antibiotic penicillin G, and the results indicated that the parts of the test plant have significant antibacterial activity, which may support traditional claims and could be candidates for alternative drug discoveries.

Neutrophil accumulation raises defence against Streptococcus equi ssp. zooepidemicus in the absence of Gasdermin D.

Streptococcus equi ssp. zooepidemicus (SEZ) predominantly acts as a zoonotic pathogen, capable of infecting a diverse range of animal species including human. Gasdermin D (GSDMD) exhibited comprehensive functions in host against different pathogenic microorganism. This study aimed to investigate the role of GSDMD in host against SEZ. Mice were administrated with SEZ via intranasal intubation for 24 h (3 × 106CFU), GSDMD protein expression significantly increased in the lung tissue of mice infected with SEZ. For further research on the role of GSDMD during SEZ infection, GSDMD-/- mice and WT mice were treated with SEZ via intranasal intubation for 24 h (3 × 106CFU). GSDMD-/- mice showed less severe lung tissue due to fewer bacteria colonization. Numerous neutrophils were recruited into lung tissues in GSDMD-/- mice, related to the release of CXCL1 and CXCL2 regulated by p65 phosphorylation. In further study, neutrophils of WT and GSDMD-/- mice were isolated and treated with SEZ (multiplicity of infection, MOI = 10, 4 h). The absence of GSDMD alleviated the death of neutrophils, in addition, GSDMD deficiency could promote translocation of p65 from the cytoplasm into the nucleus in neutrophil, which may contribute to the release of IL-1ß and TNF-α. This study demonstrated a novel function of GSDMD in host immune response to SEZ invading, indicating that GSDMD deficiency ameliorated SEZ infection through enhancing neutrophil accumulation into infected site, and activating NF-κB pathway in neutrophil to release cytokines against SEZ. Our study suggested that inhibition of host GSDMD may be an effective method against SEZ.

Magnolol attenuates macrophage pyroptosis triggered by Streptococcus equi subsp. zooepidemicus.

Streptococcus equi subsp. zooepidemicus (SEZ) is a zoonotic bacterial pathogen that causes life-threatening infections and various diseases such as meningitis, endocarditis and pneumonia. With the use of antibiotics being severely restricted in the international community, an alternative to antibiotics is urgently needed against bacterial. In the present study, the herbal extract magnolol protected mice against SEZ infection, reflected by increased survival rate and reduced bacterial burden. A pro-inflammatory form of cell death occurred in SEZ-infected macrophage. Magnolol downregulated the expression of pyroptosis-related proteins and reduced the formation of cell membrane pores in infected macrophages to suppress the development of subsequent inflammation. We further demonstrated that magnolol directly suppressed SEZ-induced macrophage pyroptosis, which partially protected macrophages from SEZ infection. Our study revealed that magnolol suppressed inflammation and protected mice against SEZ infection, providing a possible treatment for SEZ infection.

Streptococcus zooepidemicus in dogs: Exploring a canine pathogen through multilocus sequence typing.

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.

Characterization of AI-2/LuxS quorum sensing system in biofilm formation, pathogenesis of Streptococcus equi subsp. zooepidemicus.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of both humans and animals. Quorum sensing (QS) plays an important role in the regulation of bacterial group behaviors. The aim of this study was to characterize the LuxS in SEZ and evaluate its impact on biofilm formation, pathogenesis and gene expression. The wild-type SEZ and its LuxS mutant (ΔluxS) were examined for growth, biofilm formation, virulence factors, and transcriptomic profiles. Our results showed that LuxS deficiency did not affect SEZ hemolytic activity, adhesion or capsule production. For biofilm assay demonstrated that mutation in the luxS gene significantly enhances biofilm formation, produced a denser biofilm and attached to a glass surface. RAW264.7 cell infection indicated that ΔluxS promoted macrophage apoptosis and pro-inflammatory responses. In mice infection, there was no significant difference in mortality between SEZ and ΔluxS. However, the bacterial load in the spleen of mice infected with ΔluxS was significantly higher than in those infected with SEZ. And the pathological analysis further indicated that spleen damage was more severe in the ΔluxS group. Moreover, transcriptomics analysis revealed significant alterations in carbon metabolism, RNA binding and stress response genes in ΔluxS. In summary, this study provides the first evidence of AI-2/LuxS QS system in SEZ and reveals its regulatory effects on biofilm formation, pathogenicity and gene expression.

Multi-locus sequence typing and in vitro antimicrobial resistance of equine Streptococcus equi subspecies zooepidemicus strains.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the most important pathogens frequently associated with the main causes of equine infertility. In this study, we surveyed 22 strains of S. zooepidemicus collected during 2021 from cervico-uterine swabs of mares with endometritis. The genetic variability of the isolated strains was studied by multi-locus sequence typing (MLST) from whole-genome sequencing (WGS) data. The average length of reconstructed genomes was 2,088,286 bp (95% CI: 2,061,569 bp-2,114,967 bp), which was expected for S. zooepidemicus genomes. The assembled genomes were assigned to sequence types (STs) using the S. zooepidemicus scheme targeting seven loci (arcC, nrdE, proS, spi, tdk, tpi, yqiL) available in PubMLST database. MLST revealed a wide variability of STs with two (9.1%) novel STs identified in this study, precisely ST521 with two isolates and ST522 with one isolate. Furthermore, 4/22 (18.2%) isolates were assigned to ST92, 3/22 (13.6%) to ST205, 2/22 (9.1%) to ST475, and one strain (4.5%) for each of the following STs: ST10, ST30, ST39, ST49, ST101, ST132, ST147, ST314, ST369, ST467. Isolates were also tested for antimicrobial resistance using Kirby-Bauer disk diffusion method. Resistance to amoxicillin-clavulanate, ampicillin, amikacin, gentamicin, streptomycin, enrofloxacin, sulfamethoxazole-trimethoprim, tetracycline, oxytetracycline represented the most common resistance profile (13/22, 59.1%). No correlation between specific ST and antimicrobial resistance profile was found. Our study provides a comprehensive insight into the epidemiology, ST diversity and antimicrobial resistance profile of S. zooepidemicus strains, isolated in Italy, causing subfertility problems in mares.

Organ distribution and early pathogenesis of Streptococcus equi subsp. zooepidemicus in swine.

Streptococcus equi subsp. zooepidemicus is an emerging pathogen of pigs, resulting in high-mortality outbreaks of septicaemia and abortions. Here, we investigated the early pathogenesis of S. zooepidemicus in pigs following oronasal inoculation. Fourteen pigs were inoculated with live cultures of S. zooepidemicus ST-194, and monitored at 2,4, 8, and 24 h post-inoculation. Necropsies were performed to assess gross lesions and collect samples for bacterial culture and PCR analysis at each time point. Our findings revealed that S. zooepidemicus was detectable in various organs as early as 2 h post-inoculation, including liver and spleen, demonstrating rapid dissemination within the host. Tonsil samples consistently harboured live S. zooepidemicus throughout the study period, suggesting their potential for epidemiological sampling and diagnostics. Moreover, the presence of varying bacterial loads in mesenteric lymph nodes indicated persistence, replication, and a potential source for shedding. Further studies are required to determine the initial site of replication.

Dynamics analysis of strangles with asymptomatic infected horses and long-term subclinical carriers.

Strangles is one of the most prevalent horse diseases globally. The infected horses may be asymptomatic and can still carry the infectious pathogen after it recovers, which are named asymptomatic infected horses and long-term subclinical carriers, respectively. Based on these horses, this paper establishes a dynamical model to screen, measure, and model the spread of strangles. The basic reproduction number $ \mathcal{R}_0 $ is computed through a next generation matrix method. By constructing Lyapunov functions, we concluded that the disease-free equilibrium is globally asymptotically stable if $ \mathcal{R}_0 < 1 $, and the endemic equilibrium exits uniquely and is globally asymptotically stable if $ \mathcal{R}_0 > 1 $. For example, while studying a strangles outbreak of a horse farm in England in 2012, we computed an $ \mathcal{R}_0 = 0.8416 $ of this outbreak by data fitting. We further conducted a parameter sensitivity analysis of $ \mathcal{R}_0 $ and the final size by numerical simulations. The results show that the asymptomatic horses mainly influence the final size of this outbreak and that long-term carriers are connected to an increased recurrence of strangles. Moreover, in terms of the three control measures implemented to control strangles(i.e., vaccination, implementing screening regularly and isolating symptomatic horses), the result shows that screening is the most effective measurement, followed by vaccination and isolation, which can provide effective guidance for horse management.

Molecular and sequencing study and identification of novel SeM-type in beta-hemolytic streptococci involving the upper respiratory tract in Iran.

BACKGROUND: Beta-hemolytic streptococci involving the upper respiratory tract cause strangles and strangles-like diseases in horses and cause severe economic damage to the equestrian club each year. Therefore, careful epidemiological study of these bacteria, evaluation of phylogenetic connections and SeM-typing can be useful to determine the source and epidemiological characteristics of the disease outbreak. Isolates were analyzed using molecular and phylogenetic methods and to determine antibiotic resistance pattern in Iranian isolates. Molecular and phylogenetic methods were used to evaluate Iranian streptococcal isolates, and the similarity of the Iranian SeM-97 sequence with other alleles was assessed using the Neighbor-joining method with the Kimura 2 Parameter statistical model. The amino acid sequence of this gene was compared with the predicted SeM-3 reference amino acid sequence (FM204883) using MEGA 7 software. RESULTS: One type of SeM was found among streptococcal isolates. This type (SeM-97) was reported for the first time and was a new SeM. The relationship between streptococcal isolates and age, sex, race, clinical signs and geographical area was investigated. A significant relationship was observed between streptococcal isolates with age variables and clinical symptoms. CONCLUSIONS: In our study, a Streptococcus equi subsp. equi genotype was identified. The 97 allele of this gene has not been officially reported anywhere and is only registered in the Public databases for molecular typing and microbial genome diversity (PubMLST)-SeM database by Katy Webb. This was the first isolate reported and registered in the mentioned database. The isolate (Tabriz61) had the SeM-97 allele with clinical signs including mucopurulent discharge, abnormal sounds in lung hearing, warmth and enlargement or discharge and abscess of retropharyngeal lymph node and fever. This isolate was sensitive to penicillin, meropenem, ampicillin, cefotaxime, tetracycline, erythromycin, azithromycin, chloramphenicol, enrofloxacin and ciprofloxacin antibiotics and resistant to trimethoprim-sulfamethoxazole and gentamicin antibiotics.

Genomic characterization of Streptococcus equi subspecies zooepidemicus from a 2021 outbreak in Indiana with increased sow mortality.

IMPORTANCE: This study highlights a Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) strain isolated from an outbreak in Indiana, which resulted in mortality events among a swine herd in 2021. The Indiana outbreak strain was found to be genetically and phylogenetically distant to a strain isolated from the 2019 outbreaks in Ohio and Tennessee, which caused high swine mortality. We also discovered multiple unique genetic features in the Indiana outbreak strain, including distinct S. zooepidemicus genomic islands, and notable S. zooepidemicus virulence genes-many of which could serve as biomarkers for the diagnosis of this strain. These findings provide significant insights into monitoring and potentially preventing severe outbreaks caused by the Indiana outbreak strain in the future.

A case of bacteremia and pneumonia caused by Streptococcus equi subspecies equi infection in a 70-year-old female following horse exposure in rural Wyoming.

BACKGROUND: The occurrence of zoonotic infections following an animal exposure continues to be an important consideration for all patients, especially those within agricultural communities. Streptococcus equi subspecies equi (S. equi subsp. equi) is a bacteria known to cause a common infection called 'Strangles' in horses. This article highlights a new case of pneumonia and bacteremia in a patient caused by S. equi subsp. equi following strangles exposure in a horse. Rarely has there been reported horse to human transmission of subsp. equi. CASE PRESENTATION: A 70-year-old woman attended a rural emergency department with complaints of dry heaving, fever, chills, shakes, and nausea and presented with a cough. She had undergone a screening colonoscopy two days prior with no other significant medical history. The patient had computed tomography (CT) evidence of a pneumonia and positive blood cultures growing S. equi subsp. equi consistent with bacteremia. The patient later disclosed the recent passing of her horse following its sudden illness six days prior to her emergency department presentation. She had cuddled and kissed the horse prior to its death. The patient was treated with IV lactated ringers during the initial evaluation and admission and also received IV piperacillin-tazobactam 4.5 g every eight hours intravenously during her hospital stay. She was transitioned to an oral antibiotic on discharge. Subsequent blood cultures drawn the day after discharge were negative for S. equi subsp. equi, indicating successful treatment of her bacteremia. CONCLUSIONS: This report discusses an atypical presentation of S. equi subsp. equi infection in an otherwise healthy individual, manifesting as early sepsis, pneumonia, and bacteremia. The patient likely developed this infection following direct contact exposure to her horse who had died from presumed strangles a few days prior to her symptom onset. This case highlights the importance of investigating potential exposures to S. equi subsp. equi in rural areas, areas where farming and ranching are prevalent, particularly among individuals working with horses. It is especially important to acknowledge high risk populations such as immunocompromised individuals with signs and symptoms of meningitis or bacteremia.

Detection of Viable Streptococcus equi equi Using Propidium Monoazide Polymerase Chain Reaction.

There is debate around the clinical significance of Streptococcus equi subsp. equi detection in low numbers using quantitative real-time PCR (qPCR). Propidium monoazide (PMA) qPCR has been used to differentiate DNA from viable and nonviable bacterial cells. The aim of this study was to evaluate the ability of PMA eqbE SEQ2190 triplex qPCR to differentiate DNA from viable and nonviable S. equi in positive and suspect positive clinical specimens. Fifty-seven stored (frozen and refrigerated) positive (36) or suspect positive (21) clinical specimens (determined via SeeI qPCR as the gold standard) were tested using eqbE SEQ2190 triplex qPCR with (+) and without (-) PMA pretreatment. Cycle thresholds were higher when using PMA indicating a mixture of heat killed and viable cells. Number of S. equi positive specimens were as follows: 6/57 eqbE + PMA, 13/57 eqbE -PMA (Chi- squared 3.1, p = .079); 10/57 SEQ2190 +PMA, 53/57 SEQ2190 -PMA (Chi- squared 65.6, p < .0001). The mean cycle thresholds were as follows: 23.88 eqbE -PMA, 29.89 eqbE + PMA (p = .04); 24.9 SEQ2190 -PMA, 31.9 SEQ2190 +PMA (p < .0001). PMA qPCR can be used to determine S. equi viability, but testing should be performed on fresh specimens.

A distinct variant of the SzM protein of Streptococcus equi subsp. zooepidemicus recruits C1q independent of IgG binding and inhibits activation of the classical complement pathway.

Streptococcus equi subsp. zooepidemicus (SEZ) is a major equine pathogen that causes pneumonia, abortion, and polyarthritis. It can also cause invasive infections in humans. SEZ expresses the M-like protein SzM, which recruits host proteins such as fibrinogen to the bacterial surface. Equine SEZ strain C2, which binds only comparably low amounts of human fibrinogen in comparison to human SEZ strain C33, was previously shown to proliferate in equine and human blood. As the expression of SzM_C2 was necessary for survival in blood, this study investigated the working hypothesis that SzM_C2 inhibits complement activation through a mechanism other than fibrinogen and non-immune immunoglobulin binding. Loss-of-function experiments showed that SEZ C2, but not C33, binds C1q via SzM in IgG-free human plasma. Furthermore, SzM C2 expression is necessary for recruiting purified human or equine C1q to the bacterial surface. Flow cytometry analysis demonstrated that SzM expression in SEZ C2 is crucial for the significant reduction of C3b labelling in human plasma. Addition of human plasma to immobilized rSzM_C2 and immobilized aggregated IgG led to binding of C1q, but only the latter activated the complement system, as shown by the detection of C4 deposition. Complement activation induced by aggregated IgG was significantly reduced if human plasma was pre-incubated with rSzM_C2. Furthermore, rSzM_C2, but not rSzM_C33, inhibited the activation of the classical complement pathway in human plasma, as determined in an erythrocyte lysis experiment. In conclusion, the immunoglobulin-independent binding of C1q to SzM_C2 is associated with complement inhibition.

Downstream process intensification for biotechnologically generated hyaluronic acid: Purification and characterization.

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.

Metabolic labeling of hyaluronan: Biosynthesis and quantitative analysis of 13C,15N-enriched hyaluronan by NMR and MS-based methods.

Hyaluronan (HA), a member of the GAG family of glycans, has many diverse biological functions that vary a lot depending on the length of the HA chain and its concentration. A better understanding of the structure of different-sized HA at the atomic level is therefore crucial to decipher these biological functions. NMR is a method of choice for conformational studies of biomolecules, but there are limitations due to the low natural abundance of the NMR active nuclei 13C and 15N. We describe here the metabolic labeling of HA using the bacterium Streptococcus equi subsp. Zooepidemicus and the subsequent analysis by NMR and mass spectrometry. The level of 13C and 15N isotope enrichment at each position was determined quantitatively by NMR spectroscopy and was further confirmed by high-resolution mass spectrometry analysis. This study provides a valid methodological approach that can be applied to the quantitative assessment of isotopically labeled glycans and will help improve detection capabilities and facilitate future structure-function relationship analysis of complex glycans.