Background/Objectives: Strangles, caused by Streptococcus equi subspecies equi (S. equi), remains a common and severe equine infectious disease. Strangvac®, a recombinant fusion protein vaccine licenced in Europe, contains the antigens (Ag) CCE, Eq85, IdeE and a saponin adjuvant. Although its efficacy is high (94% in clinical trials and 100% in some natural outbreaks), immune correlates of protection have not been defined. This study determined the antibody (Ab) thresholds predictive of protection against clinical disease following high-dose experimental S. equi infection and the expected levels of protection at 6 and 12 months after V2. Methods: This study was a retrospective analysis of six independent double-blinded placebo-controlled experimental infection studies involving 129 ponies (80 vaccinated controls and 49 placebo controls) and a serology study (12 vaccinated ponies). Ponies received two to five vaccine doses before being experimentally challenged with S. equi strain Se4047. Ponies in the serology study were not experimentally infected. The onset of pyrexia (≥39 °C for at least 2 of 3 consecutive days, OOT) was used as a disease marker. Serology to IdeE, Eq85 and CCE was analysed with standardised clinical outcomes to define protective thresholds through correlation and Receiver Operating Characteristic (ROC) analyses. The predicted level of protection up to one year after V2 was then calculated (duration of immunity: DOI). Results: A protection threshold of ≥10 days to OOT, derived from the control distribution, was used for ROC modelling. Predictive performance (e.g., accuracy, precision, specificity) was calculated for individual and combined Ab thresholds. All controls developed pyrexia (median 6 days, IQR 5–7), with 46 out of 49 (93.9%) within 9 days of the challenge. Vaccinated ponies showed significantly delayed or absent OOT compared with controls (p < 0.0001), with 37 vaccinated ponies (46.25%) reaching the end of the studies without developing pyrexia. The Ab titre to all antigens was significantly associated with the level of protection (p < 0.0001). ROC analyses demonstrated high discriminative power (AUC 0.86–0.88). Optimal Ab titre boundaries yielded high precision (≥80%) for all Ags (IdeE: 3.5–4.3; Eq85: 2.65–3.7 and CCE: 2.66–3.2). Both precision and accuracy remained above 80% for levels of IdeE and Eq85 Ab titres superior or equal to those measured up to one year after V2, with an estimated level of protection of 78.9% to 81.2% in vaccinated animals. Conclusions: Ab titres to all three Ags represent robust correlates of protection against pyrexia following high-dose experimental S. equi challenge in Strangvac®-vaccinated ponies. Ab titres measured up to one year after V2 were estimated to continue to provide significant protection in vaccinated animals. These findings support the observed levels of protection conferred by Strangvac® against natural infection with S. equi.

BACKGROUND:Streptococcus equi subspecies zooepidemicus is a highly diverse opportunistic pathogen of horses, associated with respiratory disease and endometritis. OBJECTIVES:To characterise S. zooepidemicus isolates recovered from young ponies during a natural episode of respiratory disease, and to determine if vaccination with Strangvac®, a vaccine against Streptococcus equi subspecies equi, conferred cross-protection. STUDY DESIGN:Retrospective analysis of a double-blinded placebo-controlled study involving 32 ponies (16 vaccinates and 16 placebo controls) that experienced a natural episode of respiratory disease around the time of second vaccination. METHODS:Ponies were monitored daily for clinical signs of respiratory infection. Nasopharyngeal swabs were taken from affected animals to identify equine pathogens by qPCR and culture. The genomes of 23 S. zooepidemicus isolates were sequenced. Clinical and serological differences between vaccine and placebo groups were determined. RESULTS:The clinical samples were positive for equine herpes virus-4 or S. zooepidemicus (1/15 [7%] and 13/15 [87%], respectively). Seven different S. zooepidemicus sequence types, which encoded between four and seven of the eight antigens in Strangvac® with ≥70% amino acid identity, were recovered from affected horses. The most conserved antigens were CNE, EAG, Eq5 and IdeE. The number of days that ponies had a cough was significantly associated (p = 0.005) with the vaccination status, with fewer days in the vaccinated compared to co-mingled control ponies (1.8 ± 1.9 days vs. 3.4 ± 3.5 days). A higher antibody titre to IdeE immediately prior to second vaccination correlated with a lower cumulative coughing score (p = 0.03). A reduced number of days with abnormal temperature correlated with increased antibody levels against Eq85 (p = 0.023-0.028). MAIN LIMITATIONS:Small group size. CONCLUSIONS:This study provides evidence in support of a cross-protective effect of Strangvac® for the reduction of clinical signs associated with natural infection with S. zooepidemicus, which correlated with the presence of higher antibody titres to IdeE and Eq85.

This focus article has been prepared by Romain Paillot, Francesco Righetti, Carl Robinson, Lars Frykberg, Margareta Flock, Olof Zachrisson, Bengt Guss, Jan‐Ingmar Flock and Andrew S Waller . It reflects on a recently published study investigating correlations between strangles vaccine‐induced antibody titres and levels of protection and duration of immunity in horses.

Infection of susceptible horses with Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is associated with commingling. Exposure may occur among horses at equestrian events, sales, or horses moved among different equine stabling environments. Strangles can affect all horses on a farm, leading to the death of up to 10% of cases depending on their immunity status at the time of infection, the development of complications, the success of biosecurity measures, and the use of vaccination. The current retrospective study used ELISAs to measure the exposure of horses to S. equi at a farm that experienced an outbreak of strangles shortly after the introduction of a new horse on the same day that the majority of the 17 resident horses were vaccinated with Strangvac for the first time. One vaccinated horse, which subsequently tested positive for S. equi and EHV-4, developed a cough, elevated body temperature, and nasal discharge 11 days after the first vaccination. Two other horses developed fever for one day at 22 days post-first vaccination, but only one had serological evidence of exposure to S. equi. All vaccinated horses had high antibody titres to vaccine components, whilst 7 of the 17 resident horses, and the new arrival, tested seropositive for exposure to S. equi. Although 3 out of the 17 vaccinated horses developed mild signs of disease before second vaccination, serological data support the effectiveness of vaccination in resident populations of horses to minimise the risk of strangles following the introduction of a new horse.

Infection of horses with Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is associated with the movement of horses from one farm to another, or the attendance of equestrian events and sales. Strangles can affect all horses on a farm, leading to the death of up to 10% of cases depending on the success of biosecurity measures and the use of vaccination. This retrospective study used ELISAs to measure the exposure of horses to S. equi at a farm that experienced an outbreak of strangles shortly after the introduction of a new horse on the same day that the majority of the 17 resident horses were vaccinated with Strangvac for the first time. One vaccinated horse, which subsequently tested positive for S. equi and EHV-4, developed a cough, elevated body temperature and nasal discharge 11 days after first vaccination. Two other horses developed fever for one day at 22 days post-first vaccination, but only one had serological evidence of exposure to S. equi. All vaccinated horses had high antibody titres to vaccine components, whilst seven of the 17 resident horses, and the new arrival, tested seropositive for exposure to S. equi. Although three out of the 17 vaccinated horses developed mild signs of disease before second vaccination, serological data support the effectiveness of vaccination in resident populations of horses to minimise the risk of strangles following the introduction of a new horse.

BACKGROUND:Strangles, caused by Streptococcus equi subspecies equi (S. equi), is a prevalent infectious disease of horses. This is the first report on the use of a new vaccine, Strangvac, in a natural outbreak of strangles. OBJECTIVES:To measure the effects of Strangvac vaccination during an outbreak of strangles at a Swedish farm. STUDY DESIGN:Longitudinal cohort study. METHODS:Healthy horses (n = 17) were vaccinated on day 0, which occurred 23 days after strangles was first confirmed in three unvaccinated horses on the same farm. Blood serum samples were collected on day 0, day 28 and day 489. A combined iELISA was used to measure total antibody titres towards the components of the vaccine. Specific antibody levels in blood sera to S. equi were also quantified using the antigen A/C iELISAs, which can differentiate infected from vaccinated animals. Clinical signs were monitored. RESULTS:All vaccinated horses had increased total antibody titres to CCE, Eq85 and/or IdeE from day 0 (2.50 ± 0.28) to day 28 (3.63 ± 0.31; p < 0.0001). Seropositivity in the antigen A/C iELISA was noted in 8/17 healthy horses on day 0 (prior to vaccination), in 9/16 vaccinates on day 28, and in 3/3 unvaccinated clinical cases on day 28. None of the vaccinated horses developed strangles, but 5/7 of the original vaccinated horses tested seropositive in the A/C iELISA on day 489, which was 377 days after their third vaccination. MAIN LIMITATIONS:Aside from the three unvaccinated clinical cases, no unvaccinated control group was available. CONCLUSIONS:Despite adherence to recommended biosecurity measures, serological evidence of exposure to S. equi was demonstrated in half of the vaccinated horses. Notably, all horses vaccinated during the outbreak responded to the vaccine components and remained healthy, suggesting a protective effect of vaccination of healthy horses with Strangvac in outbreak situations.

Background: Streptococcus equi subspecies equi (S. equi) is the cause of strangles, one of the most prevalent diseases of horses worldwide. The disease is characterised by fever and the formation of abscesses in the lymph nodes of the head and neck, which can restrict the airway. A multicomponent subunit vaccine, Strangvac, has been shown to effectively reduce clinical signs of strangles and to reduce its incidence. Objective: The aim of this study was to determine the immune response against the immunoglobulin-cleaving endopeptidase IdeE, a key protective component within the vaccine and the ability of antibodies to neutralize the proteolytic activity of IdeE. Methods: An in vitro assay was developed to measure the functional inhibition of recombinant IdeE by horse sera pre- and post-vaccination. The IdeE-neutralising titres were compared to the corresponding IdeE-specific antibody titres measured by iELISA (indirect Enzyme-Linked Immunosorbent Assay). Results: A significant IdeE-specific antibody response in blood serum collected from ponies was induced after Strangvac vaccinations. Concomitantly, significant increases in the neutralising activity of IdeE occurred, persisting for at least 12 months post-second vaccination. IdeE-neutralising activity was further increased significantly after a third vaccination, even when the third dose was administered 12 months after the second dose, demonstrating that immunological memory to the vaccine persisted for 12 months. There was a significant correlation between the IdeE-neutralising activity of blood sera and the level of IdeE-specific antibodies. Conclusions: These data provide insights into one potential mechanism by which this vaccine protects Equids against or during S. equi infection.

Streptococcus suis is a severe zoonotic pathogen affecting weaned piglets. No commercial vaccine that provides protection against S. suis is available. A prototype vaccine, tentatively called VASIP (Vaccine Against Streptococcus suis Infection in Pigs), composed of five recombinant fusion proteins, encompassing 23 different protein domains, was used in this study. Pregnant sows were vaccinated on three occasions, at 68, 47 and 19 days prior to farrowing, resulting in high antibody levels, both in sera and in colostrum. Antibodies were transferred to the litter via colostrum. The litters from VASIP-vaccinated and placebo-vaccinated sows were challenged intravenously with S. suis at four or seven weeks of age in two different arms of the study. Body temperature and clinical signs (demeanour, behavioural CNS, and mobility) of infection showed that piglets from vaccinated sows were significantly protected against S. suis infection in the 4-week-old group and that the incidence of severe clinical signs was lower in the 7-week-old group compared with piglets from placebo sows. The study demonstrates the feasibility of vaccinating sows, rather than piglets, using recombinant fusion proteins to maximise protection against S. suis during the period in which they are most at risk of disease.

OBJECTIVES:The increasing frequency of antibiotic-resistant bacterial infections is a major public health challenge, and new antibiotic drugs are urgently needed. A rapid solution to the problem is to repurpose clinically approved compounds with antibacterial properties, such as the nucleoside analogues zidovudine (azidothymidine) or 5-fluoro-2'-deoxyuridine. Here we report the in vitro and in vivo antibacterial properties of double and triple combinations of azidothymidine or 5-fluoro-2'-deoxyuridine with uridine and/or trimethoprim. METHODS:We determined MICs of azidothymidine and 5-fluoro-2'-deoxyuridine, alone or combined with uridine and/or trimethoprim, against a selection of Gram-negative and Gram-positive bacteria. We also measured MICs of a selection of antibiotics of different classes as a function of uridine concentration. The efficacy of azidothymidine and 5-fluoro-2'-deoxyuridine with uridine and/or trimethoprim was measured in a murine peritonitis infection model. RESULTS:The addition of uridine enhanced the in vitro antibacterial activity of azidothymidine and 5-fluoro-2'-deoxyuridine, against Gram-negative and Gram-positive bacteria, respectively. Uridine also enhanced the in vitro antibacterial activity of azidothymidine/trimethoprim and 5-fluoro-2'-deoxyuridine/trimethoprim combinations. Triple combinations containing azidothymidine, trimethoprim and uridine, showed antibacterial synergy against Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) whereas the 5-fluoro-2'-deoxyuridine, trimethoprim and uridine combination showed synergy against the Gram-positive Staphylococcus aureus. The positive effect of uridine on the efficacy of azidothymidine/trimethoprim combination was also observed in vivo in a murine E. coli peritonitis model. CONCLUSIONS:Triple combinations of these clinically approved compounds warrant further investigations as therapies to combat antibiotic-resistant infections.

The impact of S. suis on Swedish pig production has increased in recent years, and characterization of the strains present in the pig population is needed to aid in surveillance and prevention. Therefore, the aim of this study was to identify and characterize differences in the genomes between Swedish S. suis isolates associated with disease and isolates from healthy animals. Isolates categorized as being pathogenic (n = 100) or non-pathogenic (n = 117) were whole-genome sequenced, serotyped in silico, and sequence-typed using traditional MLST and core-genome MLST, and a genome-wide association study was performed to identify virulence-associated genes. In decreasing order, serotypes 2, 1, and 7 were the most common in the pathogenic group, and serotypes 15 and 12 were the most common in the non-pathogenic group. Among the commonly disease-associated sequence types, ST28 and ST25 were identified, whereas ST1 was scarcely found. The majority of isolates belonged to novel sequence types, revealing differences between Swedish isolates and those reported from other countries. The genomes of the pathogenic isolates were on average smaller and less heterogenic as compared to those of the non-pathogenic isolates. Although a majority of the previously published virulence-associated genes included in the study were found in the genomes of both pathogenic and non-pathogenic isolates, several new, significantly virulence-associated genes were identified.

Streptococcus dysgalactiae subsp. equisimilis (Sde) is a commensal bacterium of horses that causes opportunistic infections. The aim of the work was to study genotypic and phenotypic properties of the Sde strain related to equine neonatal mastitis. Sde was isolated from an 8 day-old filly and sequenced for genome analysis, antibiotic susceptibility tests and virulence factor (VF) assays. The Sde strain presented the novel emm-subtype stC839.12 and the novel multilocus-sequence type ST-670, which belonged to a specific equine genotype group. Although no specific genotypic mechanisms related to antibiotic resistance were found, it presented genes encoding efflux pumps and transporters pmrA, bmrC and lmrP. Genes encoding several putative VFs including emm, cpa, fbp-2, adcA, hyl, htrA, tig, slo, and ndk and loci-encoding phosphoenolpyruvate-protein phosphotransferase systems were identified. This is the first report of an equine neonatal mastitis case caused by a novel genotype and horse specific Sde strain.

Summary Background Streptococcus equi subspecies equi ( S equi ) is the cause of Strangles, one of the most prevalent diseases of horses worldwide. Variation within the immunodominant SeM protein has been documented, but a new eight‐component fusion protein vaccine, Strangvac, does not contain live S equi or SeM and conservation of the antigens it contains have not been reported. Objective To define the diversity of the eight Strangvac antigens across a diverse S equi population. Study design Genomic description. Methods Antigen sequences from the genomes of 759 S equi isolates from 19 countries, recovered between 1955 and 2018, were analysed. Predicted amino acid sequences in the antigen fragments of SEQ0256(Eq5), SEQ0402(Eq8), SEQ0721(EAG), SEQ0855(SclF), SEQ0935(CNE), SEQ0999(IdeE), SEQ1817(SclI) and SEQ2101(SclC) in Strangvac and SeM were extracted from the 759 assembled genomes and compared. Results The predicted amino acid sequences of SclC, SclI and IdeE were identical across all 759 genomes. CNE was truncated in the genome of five (0.7%) isolates. SclF was absent from one genome and another encoded a single amino acid substitution. EAG was truncated in two genomes. Eq5 was truncated in four genomes and 123 genomes encoded a single amino acid substitution. Eq8 was truncated in three genomes, one genome encoded four amino acid substitutions and 398 genomes encoded a single amino acid substitution at the final amino acid of the Eq8 antigen fragment. Therefore, at least 1579 (99.9%) of 1580 amino acids in Strangvac were identical in 743 (97.9%) genomes, and all genomes encoded identical amino acid sequences for at least six of the eight Strangvac antigens. Main limitations Three hundred and seven (40.4%) isolates in this study were recovered from horses in the UK. Conclusions The predicted amino acid sequences of antigens in Strangvac were highly conserved across this collection of S equi .

Isopedopeptins are antibiotic cyclic lipodepsipeptides containing the subsequence L-Thr—L-2,3-diaminopropanoic acid—D-Phe—L-Val/L-3-hydroxyvaline. Acidic hydrolysis of isopedopeptins in D2O showed the D-Phe residues to racemize extensively in peptides with L-3-hydroxyvaline but not in peptides with L-Val. Similarly, one Leu residue in pedopeptins, which are related peptides containing the subsequence Leu—2,3-diaminopropanoic acid—Leu—L-Val/L-3-hydroxyvaline, was found to racemize in peptides with L-3-hydroxyvaline. Model tetrapeptides, L-Ala—L-Phe—L-Val/3-hydroxyvaline—L-Ala, gave the corresponding results, i.e. racemization of L-Phe only when linked to a L-3-hydroxyvaline. We propose the racemization to proceed via an oxazoline intermediate involving Phe/Leu and the L-3-hydroxyvaline residues. The 3-hydroxyvaline residue may form a stable tertiary carbocation by loss of the sidechain hydroxyl group as water after protonation. Elimination of the Phe/Leu H-2 and ring-closure from the carbonyl oxygen onto the carbocation results in the suggested oxazoline intermediate. The reversed reaction leads to either retained or inversed configuration of Phe/Leu. Such racemization during acidic hydrolysis may occur whenever a 3-hydroxyvaline residue or any amino acid that can form a stable carbocation on the C-3, is present in a peptide. The proposed mechanism for racemization was supported by incorporation of 18O in the 3-hydroxyvaline sidechain when the acidic hydrolysis was performed in H2O/H218O (1:1). The 2,3-diaminopropanoic residues of isopedopeptins and pedopeptins were also found to racemize during acidic hydrolysis, as previously described. Based on the results, the configuration of the Leu and 2,3-diaminopropanoic acid residues of the pedopeptins were reassigned to be L-Leu and D-Leu, and 2 × L-2,3-diaminopropanoic acid.

Twenty-eight multidrug-resistant bacterial strains closely related or identical to Pedobacter cryoconitis, Pedobacter lusitanus and Pedobacter steynii were isolated from soil samples by selection for multidrug-resistance. Approximately 3–30% of the selected isolates were identified as Pedobacter, whereas isolation without antibiotics did not yield any isolates of this genus. Next generation sequencing data showed Pedobacter to be on 69th place among the bacterial genera (0.32% of bacterial sequences). The Pedobacter isolates produced a wide array of novel compounds when screened by UHPLC-MS/MSMS, and hierarchical cluster analysis resulted in several distinct clusters of compounds produced by specific isolates of Pedobacter, and most of these compounds were found to be peptides. The Pedobacter strain UP508 produced isopedopeptins, whereas another set of strains produced pedopeptins, which both are known cyclic lipodepsipeptides produced by Pedobacter sp. Other Pedobacter strains produced analogous peptides with a sequence variation. Further strains of Pedobacter produced additional novel antibacterial cyclic lipopeptides (ca 800 or 1400 Da in size) and/or linear lipopeptides (ca 700–960 Da in size). A 16S rRNA phylogenetic tree for the Pedobacter isolates revealed several distinct clades and subclades of isolates. One of the subclades comprised isolates producing isopedopeptin analogs, but the isopedopeptin producing isolate UP508 was clearly placed on a separate branch. We suggest that the non-ribosomal peptide synthases producing pedopeptins, isopedopeptins, and the analogous peptides, may derive from a common ancestral non-ribosomal peptide synthase gene cluster, which may have been subjected to a mutation leading to changed specificity in one of the modules and then to a modular rearrangement leading to the changed sequence found in the isopedopeptins produced by isolate UP508.

The equine disease strangles, which is characterized by the formation of abscesses in the lymph nodes of the head and neck, is one of the most frequently diagnosed infectious diseases of horses around the world. The causal agent, Streptococcus equi subspecies equi, establishes a persistent infection in approximately 10 % of animals that recover from the acute disease. Such 'carrier' animals appear healthy and are rarely identified during routine veterinary examinations pre-purchase or transit, but can transmit S. equi to naïve animals initiating new episodes of disease. Here, we report the analysis and visualization of phylogenomic and epidemiological data for 670 isolates of S. equi recovered from 19 different countries using a new core-genome multilocus sequence typing (cgMLST) web bioresource. Genetic relationships among all 670 S. equi isolates were determined at high resolution, revealing national and international transmission events that drive this endemic disease in horse populations throughout the world. Our data argue for the recognition of the international importance of strangles by the Office International des Épizooties to highlight the health, welfare and economic cost of this disease. The Pathogenwatch cgMLST web bioresource described herein is available for tailored genomic analysis of populations of S. equi and its close relative S. equi subspecies zooepidemicus that are recovered from horses and other animals, including humans, throughout the world. This article contains data hosted by Microreact.

Streptococcus suis is an important bacterial pathogen in pigs that may also cause zoonotic disease in humans. The aim of the study was to evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification of S. suis case isolates from diseased pigs and tonsil isolates from healthy pigs and wild boar using sequence analysis methods. Isolates (n = 348) that had been classified as S. suis by MALDI-TOF MS were whole-genome sequenced and investigated using analyses of (i) the 16S rRNA gene, (ii) the recN gene, and (iii) whole-genome average nucleotide identity (ANI). Analysis of the 16S rRNA gene indicated that 82.8% (288 out of 348) of the isolates were S. suis, while recN gene analysis indicated that 75.6% (263 out of 348) were S. suis. ANI analysis classified 44.3% (154 out of 348) as S. suis. In total, 44% (153 out of 348) of the investigated isolates were classified as S. suis by all of the species identification methods employed. The mean MALDI-TOF MS score was significantly higher for the S. suis case isolates than for the tonsil isolates; however, the difference is of limited practical use. The results show that species confirmation beyond MALDI-TOF MS is needed for S. suis isolates. Since the resolution of 16S rRNA gene analysis is too low for Streptococcus spp., ANI analysis with a slightly lowered cutoff of 94% may be used instead of, or in addition to, recN gene analysis. Supplementation of the MALDI-TOF MS reference library with mass spectra from S. orisratti, S. parasuis, S. ruminantium, and additional S. suis serotypes should be considered in order to produce more accurate classifications.

In the search for new antibiotic compounds, fractionation of Pseudomonas protegens UP46 culture extracts afforded several known Pseudomonas compounds, including 2,4-diacetylphloroglucinol (DAPG), as well as two new antibacterial alkaloids, 6-(pyrrolidin-2-yl)DAPG ( 1 ) and 6-(piperidin-2-yl)DAPG ( 2 ). The structures of 1 and 2 were determined by nuclear magnetic resonance spectroscopy and mass spectrometry. Compounds 1 and 2 were found to have antibacterial activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus , with minimal inhibitory concentration (MIC) 2 and 4 μg ml −1 , respectively, for 1 , and 2 μg ml −1 for both pathogens for 2 . The MICs for 1 and 2 , against all tested Gram-negative bacteria, were >32 μg ml −1 . The half maximal inhibitory concentrations against HepG2 cells for compounds 1 and 2 were 11 and 18 μg ml −1 , respectively, which suggested 1 and 2 be too toxic for further evaluation as possible new antibacterial drugs. Stable isotope labelling experiments showed the pyrrolidinyl group of 1 to originate from ornithine and the piperidinyl group of 2 to originate from lysine. The P. protegens acetyl transferase (PpATase) is involved in the biosynthesis of monoacetylphloroglucinol and DAPG. No optical rotation was detected for 1 or 2 , and a possible reason for this was investigated by studying if the PpATase may catalyse a stereo-non-specific introduction of the pyrrolidinyl/piperidinyl group in 1 and 2 , but unless the PpATase can be subjected to major conformational changes, the enzyme cannot be involved in this reaction. The PpATase is, however, likely to catalyse the formation of 2,4,6-triacetylphloroglucinol from DAPG.

The equine disease strangles, caused by Streptococcus equi, remains a major cause of welfare and economic cost to the global horse industry. Here we report the safety, immunogenicity and efficacy of a novel multi-component chimeric fusion protein vaccine, called Strangvac, when administered to ponies via the intramuscular route. Across the four studies, Strangvac was safe and induced robust antibody responses towards the vaccine components in blood serum and the nasopharynx, which were boosted by revaccination up to 12 months after a primary course of 2 vaccinations 4 weeks apart. The vaccine response did not cross-react with a commercial strangles iELISA, which identifies horses that have been exposed to S. equi, demonstrating that it was possible to differentiate infected from vaccinated animals (DIVA). Following challenge with S. equi strain 4047 (Se4047), all 36 control ponies that had received an adjuvant-only placebo vaccine developed clinical signs of strangles. In contrast, intramuscular vaccination with Strangvac protected ponies significantly from challenge with Se4047 at two weeks (5 of 16 ponies protected (31%), P = 0.04) and two months (7 of 12 ponies protected (58%), P = 0.0046 (including pooled control data) after second vaccination. Optimal protection (15 of 16 ponies protected (94%), P < 0.0001) was observed following challenge at two weeks post-third vaccination. Our data demonstrate that Strangvac is safe, has DIVA capability and provides a rapid onset of protective immunity against strangles. We conclude that Strangvac is a valuable tool with which to protect horses from strangles, particularly during high-risk periods, whilst maintaining the mobility of horse populations as required by the global equine industry. (C) 2020 The Authors. Published by Elsevier Ltd.

Pedobacter cryoconitis strain UP508 was isolated from a soil sample using a mixture of ampicillin, kanamycin, and nalidixic acid for selection. UP508 was found to produce >30 unknown antibacterial peptides, of which eight, isopedopeptins A-H (1-8), were isolated by bioassay-guided fractionation and characterized with respect to structures and biological properties. Compounds 1-8 were all composed of nine amino acid residues and one 3-hydroxy fatty acid residue, and the structures were ring-closed via an ester bond from the C-terminal aspartic acid to the 3-hydroxy fatty acid. The differences between the peptides were the size and branching of the 3-hydroxy fatty acid and the presence of a valine or a 3-hydroxyvaline residue. The isopedopeptins mainly had activity against Gram-negative bacteria, and isopedopeptin B (2), which had the best combination of antibacterial activity, in vitro cytotoxicity, and hemolytic properties, was selected for further studies against a larger panel of Gram-negative bacteria. Isopedopeptin B was found to have good activity against strains of WHO top-priority Gram-negative bacteria, i.e., carbapenem-resistant Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, with minimal inhibitory concentrations (MIC) down to 1, 2, and 4 μg/mL, respectively. Furthermore, compound 2 had activity against colistin-resistant strains of A. baumannii, E. coli, and Klebsiella pneumoniae, with a MIC down to 8, 2, and 4 μg/mL, respectively. Compound 6 was tested in an E. coli liposome system where it induced significant leakage, indicating membrane disruption as one mechanism involved in isopedopeptin antibacterial activity. Isopedopeptin B stands out as a promising candidate for further studies with the goal to develop a new antibiotic drug.