Phenotypic, molecular and genomic characterization of Actinobaculum suis isolated from swine in Brazil.

Urinary tract infections (UTI) are considered one of the most important diseases of sows due to its close relationship with reproductive problems such as reduced litter size, increase in the rate of return to estrous, vulvar discharge, abortion, mastitis and anestrus. Actinobaculum suis is one of the main agents involved in porcine urinary tract infection and is responsible for the most severe and fatal cases in sows. In the present report, 23 A. suis strains isolated from a sow and boars in Brazil were identified by PCR and further characterized by broth microdilution, molecular typing by pulsed-field gel electrophoresis (PFGE), single-enzyme amplified fragment length polymorphism (SE-AFLP), and whole-genome sequencing. All strains were sensitive to ceftiofur, linezolid, nitrofurantoin, quinupristin-dalfopristin and vancomycin. Ciprofloxacin, daptomycin, lincomycin, erythromycin and tylosin resistance was observed in 100% of tested strains. Tetracycline and tigecycline also presented high resistance rates (87% and 30.4%, respectively). PFGE with eight different restriction enzymes and three programs did not enable strain characterization; however, all strains were typed by SE-AFLP that clustered strains according to their origin, thus proving an effective tool for A. suis genotyping. Whole-genome sequencing and comparative analysis enabled species differentiation from closely related genus. This is the first report of genomic characterization of A. suis.

Pathogenicity and Virulence of Trueperella pyogenes: A Review.

Bacteria from the species Trueperella pyogenes are a part of the biota of skin and mucous membranes of the upper respiratory, gastrointestinal, or urogenital tracts of animals, but also, opportunistic pathogens. T. pyogenes causes a variety of purulent infections, such as metritis, mastitis, pneumonia, and abscesses, which, in livestock breeding, generate significant economic losses. Although this species has been known for a long time, many questions concerning the mechanisms of infection pathogenesis, as well as reservoirs and routes of transmission of bacteria, remain poorly understood. Pyolysin is a major known virulence factor of T. pyogenes that belongs to the family of cholesterol-dependent cytolysins. Its cytolytic activity is associated with transmembrane pore formation. Other putative virulence factors, including neuraminidases, extracellular matrix-binding proteins, fimbriae, and biofilm formation ability, contribute to the adhesion and colonization of the host tissues. However, data about the pathogen-host interactions that may be involved in the development of T. pyogenes infection are still limited. The aim of this review is to present the current knowledge about the pathogenic potential and virulence of T. pyogenes.

Fecal microbiome signatures are different in food-allergic children compared to siblings and healthy children.

BACKGROUND: Intestinal microbes have been shown to influence predisposition to atopic disease, including food allergy. The intestinal microbiome of food-allergic children may differ in significant ways from genetically similar non-allergic children and age-matched controls. The aim was to characterize fecal microbiomes to identify taxa that may influence the expression of food allergy. METHODS: Stool samples were collected from children with IgE-mediated food allergies, siblings without food allergy, and non-allergic controls. Stool microbiome characterization was performed via next-generation sequencing (Illumina) of the V1V3 and V4 variable regions of the 16S rRNA gene. Bacterial diversity, evenness, richness, and relative abundance of the operational taxonomic units (OTUs) were evaluated using QIIME. ANOVA and Welch's t test were utilized to compare groups. RESULTS: Sixty-eight children were included: food-allergic (n = 22), non-food-allergic siblings (n = 25), and controls (n = 21). When comparing fecal microbial communities across groups, differences were noted in Rikenellaceae (P = .035), Actinomycetaceae (P = .043), and Pasteurellaceae (P = .018), and nine other distinct OTUs. Food-allergic subjects had enrichment for specific microbes within the Clostridia class and Firmicutes phylum (Oscillobacter valericigenes, Lachnoclostridium bolteae, Faecalibacterium sp.) compared to siblings and controls. Identification of Clostridium sp. OTUs revealed differences in specific Clostridia drive the separation of the allergic from the siblings and controls. Alistipes sp. were enriched in non-allergic siblings. CONCLUSIONS: Comparisons in the fecal microbiome of food-allergic children, siblings, and healthy children point to key differences in microbiome signatures, suggesting the role of both genetic and environmental contributors in the manifestation of food-allergic disease.

Imaging diagnosis--Conventional and functional magnetic resonance imaging of a brain abscess in a goat.

A 2-month-old female goat was presented for depressed mental status and multifocal central neurologic signs 3 weeks after hot-iron disbudding. Conventional magnetic resonance imaging (MRI) findings included a large intra axial mass in the left frontal lobe that was T2 hyperintense and T1 hypointense centrally with a contrast-enhancing peripheral capsule and perilesional T2 hyperintensity. A restrictive pattern was present in diffusion-weighted imaging. Magnetic resonance spectroscopy demonstrated an increased amount of succinate, acetate, amino acids, lipids; minimal amounts of lactate; and decreased amounts of N-acetyl aspartate and choline. A cerebral abscess due to Trueperella pyogenes was confirmed from necropsy and tissue culture.

Virulence determinants and biofilm production among Trueperella pyogenes recovered from abscesses of captive forest musk deer.

Trueperella pyogenes (formerly Arcanobacterium) is commonly isolated from domesticated or wild ruminants as an opportunistic pathogen. To investigate the role of virulence determinants (VDs) and biofilm production in T. pyogenes isolates, a total of 36 T. pyogenes were collected from abscesses of forest musk deer in Miyaluo Farm (Sichuan Province, China). The prevalence of VDs and associations with clonal types, antibiotic resistance and biofilm production were analyzed by PCR and bioassay. Finally, T. pyogenes isolates were separated into three clonal types based on the DNA fingerprinting of BOX-PCR. Isolates with less VDs obtained from sick forest musk deer were mainly belonged to Type 1, and the isolates with robust VD repertoire obtained from dead forest musk deer were included in Type 3. Accordingly, resistant isolates exhibited significant lower virulence than susceptible ones. Majority of T. pyogenes isolates of this study were capable of producing a biofilm. However, no VDs presence and antibiotic resistance were statistically associated with biofilm production. In conclusion, the current study demonstrated that T. pyogenes was probably the primary pathogen of abscesses in the forest musk deer. Moreover, as an animal origin pathogen, the increasing resistance of T. pyogenes isolates could also associate with a decreased virulence.

Involvement of the E3 ubiquitin ligase Cblb in host defense and evaluation of transcriptome during Trueperella pyogenes infection.

Trueperella pyogenes (T. pyogenes) is a versatile and ingenious bacterium that causes severe suppurative injuries in lots of economically important ruminants. The underlying pathogenesis of T. pyogenes infection remains poorly understood. In the current study, we performed transcriptome sequencing of mouse blood tissue infected with T. pyogenes. A total of 36.73 G clean data were collected, and 136 differentially expressed genes were obtained in the infection group compared to the control group. In addition, we found that the E3 ubiquitin ligase Cblb exhibited significant upregulation in the infection groups compared to the control group. Mechanistically, T. pyogenes infection markedly enhanced the expression of Cblb and regulated the host defense response. Inhibiting Cblb expression with Cblb siRNA impaired the inflammatory response and reduced the effect of phagocytosis in RAW264.7 murine macrophages. Intriguingly, overexpression of Cblb induced a strong inflammatory response and enhanced phagocytosis against T. pyogenes infection in macrophages. More importantly, the overexpression of Cblb significantly reduced the bacterial load and protected mice from the T. pyogenes infections. Therefore, our findings reveal that Cblb is a novel and potential regulator in response to T. pyogenes infection and shed new light on the development of promising treatments against T. pyogenes-related diseases.

An Uncommon Case of Trueperella pyogenes Infection in an Adult Backyard Rooster and a Retrospective Study; 2000-20.

Trueperella pyogenes is an opportunistic Gram-positive bacterium that induces purulent lesions and abscesses in cattle, small ruminants, and swine. In birds, T. pyogenes infections have been linked to lameness and osteomyelitis in turkeys (Phasianidae) and hepatic fibriscess in turkeys and pigeons (Columbidae). An 18-mo-old backyard rooster with a history of progressive emaciation was submitted to the California Animal Health and Food Safety (CAHFS) laboratory system. At necropsy, unusual numerous miliary granulomas were identified, primarily in the spleen, but granulomas were also observed in air sacs and lungs. Microscopically, few to moderate numbers of granulomas with giant cells were observed in the spleen, lung, air sacs, and crop composed of necrosis and mixed inflammatory cell inflammation including multinucleated giant cells, fibrin deposition, and fibrosis. Trueperella pyogenes was isolated from the air sacs and trachea. Avibacterium paragallinarum PCR was positive from the tracheal swab. A retrospective analysis of CAHFS data on T. pyogenes between 2000 and 2020 identified 24 cases in avian species: chickens (Gallus gallus domesticus; 16/24), turkeys (5/24), Pekin duck (Anas platyrhynchos domesticus; 1/24), parrot (Psittaciformes; 1/24), and pheasant (Phasianidae; 1/24). Although T. pyogenes infection in birds is rare, the clinical signs and gross lesions might be indistinguishable from avian mycobacteriosis in some cases and should be considered in the differential diagnosis.

Reporte de caso­Un caso no común de infección por Trueperella pyogenes en un gallo adulto de traspatio y un estudio retrospectivo; entre los años 2000-20. Trueperella pyogenes es una bacteria grampositiva oportunista que induce lesiones purulentas y abscesos en bovinos, pequeños rumiantes y porcinos. En las aves, las infecciones por T. pyogenes se han relacionado con cojera y osteomielitis en pavos (Phasianidae) y fibrosis hepática en pavos y palomas (Columbidae). Un gallo de traspatio de 18 meses de edad con antecedentes de emaciación progresiva fue enviado al sistema de Laboratorios de Salud Animal y Seguridad Alimentaria de California (CAHFS). En la necropsia, se identificaron numerosos granulomas miliares inusuales, principalmente en el bazo, pero también se observaron granulomas en los sacos aéreos y los pulmones. Microscópicamente, se observaron pocos a moderados granulomas con células gigantes en el bazo, pulmón, sacos aéreos y buche compuesto por necrosis e inflamación celular inflamatoria mixta, incluidas células gigantes multinucleadas, depósito de fibrina y fibrosis. Trueperella pyogenes se aisló de los sacos aéreos y la tráquea. Un método de PCR para Avibacterium paragallinarum fue positivo realizado a partir de hisopos traqueales. Un análisis retrospectivo de los datos de CAHFS sobre T. pyogenes entre los años 2000 y 2020 identificó 24 casos en especies aviares: pollos (Gallus gallus domesticus; 16/24), pavos (5/24), pato Pekín (Anas platyrhynchos domesticus; 1/24), loro (Psittaciformes; 1/24) y faisán (Phasianidae; 1/24). Aunque la infección por T. pyogenes en aves es poco común, los signos clínicos y las lesiones macroscópicas pueden ser indistinguibles de micobacteriosis aviar en algunos casos y debe considerarse como diagnóstico diferencial.

Comparison of different culture media and growth conditions for recognition of Arcanobacterium haemolyticum.

Detection of Arcanobacterium haemolyticum is based upon typical beta-hemolysis and colony morphology, but it may go undetected if only conventional sheep blood agar media for detection of beta-hemolytic streptococci are used. The influence of different culture media, atmospheres, and times of incubation for the recognition of colonies of 47 isolates of A. haemolyticum was studied. After 48 h of incubation, trypticase soy agar with 5% horse blood in 5% CO(2) was the best medium.

Influence of experimental intrauterine infusion of Arcanobacterium pyogenes solution on ovarian activity in cycling cows.

To examine the ovarian response to Arcanobacterium pyogenes (A. pyogenes) in uterus, bacterial solution was infused into the uteri of cows, and the follicle and corpus luteum (CL) development were monitored with a real-time ultrasound instrument. In addition, the plasma concentrations of progesterone (P(4)) and 13, 14-dihydro-15-keto-PGF(2alpha) (PGFM) were determined. A 10 ml bacterial solution that contained A. pyogenes (8 to 15 x 10(8) CFU/ml) was infused into the uterus of eight cows transcervically three days after natural ovulation. As a control, sterile physiological saline was infused into 4 other cows. The dominant follicle developed normally in 8 cows after bacteria inoculation. In 4 of these 8 cows, the developing CL regressed, and the first wave dominant follicles, which normally become atretic, ovulated after the inoculation. In the remaining 4 cows, the CL did not regress. The PGFM concentration increased transiently in all 8 cows after the infusion. The mean PGFM concentration of the cows with a regressed CL was significantly lower (P<0.01) than that of the cows whose CLs did not regress. In the control cows, there was no regression of developing CLs, no ovulation of first wave dominant follicles and no transient increase in PGFM after the infusion of sterile physiological saline. These results show that infusion of A. pyogenes into the uterus did not affect folliculogenesis and might have induced PGF(2alpha) production from the uterus.

Quorum-sensing molecules N-acyl homoserine lactones inhibit Trueperella pyogenes infection in mouse model.

Trueperella pyogenes is a gram-positive opportunistic pathogen normally causes mastitis, liver abscesses and pneumonia of economically important livestock. It has been suggested that gram-negative bacteria can suppress the growth and virulence of T. pyogenes in vitro by using the quorum-sensing (QS) signal molecules and cause the transition of predominant species. However, whether these QS signals can be used as potential anti-virulence drugs against T. pyogenes infection is unclear. In this study, the in vivo inhibitory effect N-acyl homoserine lactones (AHLs) from Escherichia coli and Pseudomonas aeruginosa on T. pyogenes was tested by using mouse model. Mice were first peritoneally infected with T. pyogenes followed by intravenous injection of N-Octanoyl-DL-homoserine lactone (C8HSL) or N-(3-oxododecanoyl) homoserine-l-lactone (C12HSL). The results showed that C8HSL and C12HSL significantly reduced bacterial load and increased the survival rate of mice against T. pyogenes challenge. Additionally, the treatment of AHLs promoted the secretion of IL-1ß, IL-6, IL-8 and TNF-α in mouse peritoneal fluid, and significantly decreased the expression levels of virulence genes of residual T. pyogenes. Importantly, murine macrophages rapidly phagocytosed bacteria when they were treated with AHLs compared to untreated cells. Collectively, our findings provide a major advance in understanding the inhibitory effect of AHLs in vivo and a promise for developing new clinical or veterinary treatments of T. pyogenes-related infection.

Trueperella pyogenes isolated from a brain abscess of an adult roebuck (Capreolus capreolus).

The present study was designed to characterize phenotypically and genotypically a Trueperella pyogenes strain isolated from a brain abscess of an adult roebuck (Capreolus capreolus). The species identity could be confirmed by phenotypical investigations, by MALDI-TOF MS analysis, and by sequencing the 16S ribosomal RNA (rRNA) gene, the 16S-23S rRNA intergenic spacer region (ISR); by sequencing the target genes rpoB, gap, and tuf; and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor-encoding genes which revealed the presence of the genes plo encoding pyolysin and nanH and nanP encoding neuraminidases; the genes fimA, fimC, and fimE encoding the fimbrial subunits FimA, FimC, and FimE; and the gene cbpA encoding collagen-binding protein CbpA. The present data give a detailed characterization of a T. pyogenes strain isolated from a brain abscess of a roebuck. However, the route of infection of the roebuck remains unclear.

Different inflammatory responses of bovine oviductal epithelial cells in vitro to bacterial species with distinct pathogenicity characteristics and passage number.

The bovine oviduct provides the site for fertilization and early embryonic development. Modifications to this physiological environment, for instance the presence of pathogenic bacterial species, could diminish reproductive success at early stages of pregnancy. The aim of this study was to elucidate the inflammatory responses of bovine oviductal epithelial cells (BOEC) to a pathogenic bacterial species (Trueperella pyogenes) and a potentially pathogenic bacterium (Bacillus pumilus). BOEC from four healthy animals were isolated, cultured in passage 0 (P0) and passaged until P3. Trypan blue staining determined BOEC viability during 24 h co-culture with different multiplicities of infection (MOI) of T. pyogenes (MOI 0.01, 0.05, 0.1 and 1) or B. pumilus (MOI 1 and 10). BOEC remained viable when co-cultured with T. pyogenes at MOI 0.01 and with B. pumilus at MOI 1 and 10. Extracted total RNA from control and bacteria co-cultured samples was subjected to reverse transcription-quantitative polymerase chain reaction (RTq-PCR) to determine mRNA expression of various studied genes. The rate of release of interleukin 8 (IL8) and prostaglandin E2 (PGE2) from BOEC was measured by ELISA after 24 h co-culture with bacteria. RT-qPCR of various selected pro-inflammatory factors revealed similar mRNA expression of pro-inflammatory factors in BOEC co-cultured with T. pyogenes and in the controls. Higher mRNA expression of IL 1A, -1B, tumor necrosis factor alpha and CXC ligand (CXCL) 1/2, -3, -5 and IL8 and PG synthesis enzymes in BOEC co-cultured with B. pumilus was observed. In the presence of B. pumilus a higher amount of IL8 and PGE2 was released from BOEC than from controls. The viability and pro-inflammatory response of P3 BOEC incubated with bacteria was lower than in P0 BOEC. These findings illustrate the pathogenicity of T. pyogenes towards BOEC in detail and the potential role of B. pumilus in generating inflammation in oviductal cells. Culturing conditions influenced the pro-inflammatory responses of BOEC towards bacteria. Therefore, researchers conducting epithelial-bacterial in vitro co-culture should not underestimate the effects of these parameters.

The effects of Arcanobacterium pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo.

Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F2alpha (PGF) or prostaglandin E(2) (PGE) from endometrial explants, or purified populations of endometrial epithelial or stromal cells. However, the explants produced more PGF and PGE than controls when treated with a bacteria-free filtrate (BFF) cultured from A. pyogenes. Similarly, BFF stimulated PGF and PGE production by epithelial and stromal cells, respectively. So, BFF or control PBS was infused into the uterus of heifers (n=7 per group) for 8 days, starting the day after estrus. Emergence of the follicle wave, dominant follicle or corpus luteum diameter, and peripheral plasma FSH, LH, estradiol, progesterone, PGFM, or acute phase protein concentrations were unaffected by the BFF infusion. In the live animal it is likely that the intact uterine mucosa limits the exposure of the endometrial cells to the exotoxin of A. pyogenes, whereas the cells are readily exposed to the toxin in vitro.

Biochemical changes in Bifidobacterium bifidum var. pennsylvanicus after cell wall inhibition. IX. Metabolism and release of cellular lipids in the presence of antibiotics.

Inhibiton of cell wall synthesis caused simultaneously an increase in cellular phospho-and glycolipids and a marked release of these compounds to the medium. The composition of the cellular and the released glyco-and phospholipids was almost the same. Antibiotics, which inhibit cell wall synthesis, did not influence glycolipid composition, but increased the relative and absolute amounts of disphosphatidylglycerol and its lysoderivatives. Incorporation and chase experiments demonstrated a considerable stimulation of phospholipid metabolism, and of diphosphatidylglycerol synthesis especially. Release of lipids was not accompanied by loss of cellular protein. Omission of Tween 80 from the medium decreased the release by about 50% and increased the relative amounts of the phosphogalactolipids in the cells and in the culture fluid. Inhibitors of protein synthesis and valinomycin caused a decrease in cellular lipidphosphorus content, and a relative increase of the phosphogalactolipids. No release of lipids was observed under these conditions.

Epethelial presence of Trueperella pyogenes predicts site-level presence of cranial abscess disease in white-tailed deer (Odocoileus virginianus).

Cranial/intracranial abscess disease is an emerging source of significant mortality for male white-tailed deer (Odocoileus virginianus). Most cases of cranial/intracranial abscess disease are associated with infection by the opportunistic pathogen Trueperella pyogenes although the relationship between the prevalence of the bacteria and occurrence of disease is speculative. We examined 5,612 hunter-harvested deer from 29 sites across all physiographic provinces in Georgia for evidence of cranial abscess disease and sampled the forehead, lingual, and nasal surfaces from 692 deer. We used polymerase chain reaction (PCR) to determine presence of T. pyogenes from these samples. We found T. pyogenes prevalence at a site was a predictor for the occurrence of cranial abscess disease. Prevalence of T. pyogenes did not differ between samples from the nose or tongue although prevalence along the forehead was greater for males than females (p = 0.04), particularly at sites with high occurrence of this disease. Socio-sexual behaviors, bacterial prevalence, or physiological characteristics may predispose male deer to intracranial/cranial abscess disease. Determination of factors that affect T. pyogenes prevalence among sites may help explain the occurrence of this disease among populations.

Proteolipid and calculus matrix calcification in vitro.

The initiator of calculus matrix calcification, in vitro, was isolated. Crude phospholipid, known to contain the factor, was separated into five fractions by column chromatography. A single protein-containing fraction induced apatite formation during incubation. The nucleating fraction was indentified as a proteolipid.

Comparison of calcification between Bacterionema matruchotii and Actinomyces naeslundii.

The purpose of this research was to examine the requirements for proteolipid initiation of calcification in culture. Proteolipid from a calcifiable microorganism, Bacterionema matruchotii, was compared with proteolipid isolated from a non-calcifiable microorganism, Actinomyces naeslundii. Although A. naeslundii does not calcify in culture, lyophilized cells and proteolipid-containing extracts do initiate apatite formation. A. naeslundii proteolipid (ANN) differs from B. matruchotii (BMN) in concentration, apoprotein polarity, and phospholipid composition. These differences may alter the ability of ANN to nucleate apatite in the intact cell.

Characterization of calculus matrix calcification nucleator.

The nucleator of dental calculus matrix calcification, in vitro, was analyzed. Attention focused on proteolipid singularity, amino acid composition and related polarity, and phospholipid components. The data were compared to those of the nucleator of Bacterionema matruchotii calcification.

Characterization of Bacterionema matruchotii calcification nucleator.

The nucleator of Bacterionema matruchotii calcification was characterized. Parameters examined were: proteolipid purity and singularity, amino acid composition and relative polarity, phospholipid composition, apoprotein homogeneity, essentiality of the complex for nucleation, and ordered structure. The data fulfill a requirement for comparisons among apatite-nucleating proteolipids.

Effect of viability of actinomycete spores on their ability to stimulate production of nitric oxide and reactive oxygen species in RAW264.7 macrophages.

Spores of actinomycetes, mesophilic gram-positive bacteria, isolated from moldy houses, induced the expression of inducible NO-synthase (iNOS) with a subsequent NO-production in RAW264.7 macrophages. No differences were detected between production of nitric oxide (NO) by alive or irradiated spores of different strains of Actinomycetes sp. or Streptomyces sp. Moreover, a significant production of reactive oxygen species (ROS) occurred in the macrophages after their stimulation both by alive and irradiation killed spores of actinomycetes. However, ROS-responses in macrophage induced by dead spores were significantly lower compared to those induced by alive spores. The cytotoxicity of the spores of different actinomycetes differed widely. The production of NO and ROS did not depend directly on the viability of the spores, suggesting an important role for cell wall components in the activation of the cells.