Fitness Trade-Offs between Phage and Antibiotic Sensitivity in Phage-Resistant Variants: Molecular Action and Insights into Clinical Applications for Phage Therapy.

In recent decades, phage therapy has been overshadowed by the widespread use of antibiotics in Western countries. However, it has been revitalized as a powerful approach due to the increasing prevalence of antimicrobial-resistant bacteria. Although bacterial resistance to phages has been reported in clinical cases, recent studies on the fitness trade-offs between phage and antibiotic resistance have revealed new avenues in the field of phage therapy. This strategy aims to restore the antibiotic susceptibility of antimicrobial-resistant bacteria, even if phage-resistant variants develop. Here, we summarize the basic virological properties of phages and their applications within the context of antimicrobial resistance. In addition, we review the occurrence of phage resistance in clinical cases, and examine fitness trade-offs between phage and antibiotic sensitivity, exploring the potential of an evolutionary fitness cost as a countermeasure against phage resistance in therapy. Finally, we discuss future strategies and directions for phage-based therapy from the aspect of fitness trade-offs. This approach is expected to provide robust options when combined with antibiotics in this era of phage 're'-discovery.

Innate immune response in bovine neutrophils stimulated with Mycoplasma bovis.

Mycoplasma bovis (M. bovis) is a significant worldwide pathogen of cattle. Neutrophils have an important role in the innate immune response during infection with M. bovis. However, even though neutrophils accumulate in M. bovis infection, the interaction of M. bovis and neutrophils has not been fully elucidated. We attempted to elucidate the innate immune response of neutrophils stimulated with M. bovis and evaluate the transcriptome and functional analysis of bovine neutrophils stimulated with M. bovis. Proinflammatory cytokines, such as inducible nitric oxide (iNOS), which was the most increased gene in transcriptome analysis, were increased in quantitative polymerase chain reaction analysis of bovine neutrophils stimulated with live or heat-killed M. bovis. Nitric oxide and intracellular reactive oxygen species production of neutrophils stimulated with M. bovis was significantly increased. Neutrophils stimulated with M. bovis showed an increased ratio of nonapoptotic cell death compared to unstimulated controls. We demonstrated that neutrophil extracellular traps (NETs) formation was not recognized in neutrophils stimulated with live M. bovis. However, heat-killed M. bovis induced NETs formation. We also showed the interaction with M. bovis and bovine neutrophils regarding proinflammatory cytokine gene expression and functional expression related to NETs formation. Live and killed M. bovis induced innate immune responses in neutrophils and had the potential to induce NETs formation, but live M. bovis escaped NETs.

Immunosuppression in Cows following Intramammary Infusion of Mycoplasma bovis.

Mycoplasma bovis is a destructive pathogen that causes large economic losses in rearing cattle for beef and dairy worldwide. M. bovis causes suppression of and evades the host immune response; however, the mechanisms of host immune function involved in M. bovis mastitis have not been elucidated. The purpose of this study was to elucidate the characteristics of the bovine immune response to mycoplasmal mastitis. We evaluated the responsiveness of the bovine mammary gland following infusion of M. bovis Somatic cell counts and bacterial counts in milk from the infected quarter were increased. However, the proliferation of peripheral blood mononuclear cells (blood MNCs) and mononuclear cells isolated from M. bovis-stimulated mammary lymph nodes (lymph node MNCs) did not differ from that in the unstimulated cells. Transcriptome analysis revealed that the mRNA levels of innate immune system-related genes in blood MNCs, complement factor D (CFD), ficolin 1 (FCN1), and tumor necrosis factor superfamily member 13 (TNFSF13) decreased following intramammary infusion of M. bovis The mRNA levels of immune exhaustion-related genes, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) of milk mononuclear cells (milk MNCs) in the infected quarter were increased compared with those before infusion. Increase in immune exhaustion-related gene expression and decrease in innate immune response-related genes of MNCs in quarters from cows were newly characterized by M. bovis-induced mastitis. These results suggested that M. bovis-induced mastitis affected the immune function of bovine MNCs, which is associated with prolonged duration of infection with M. bovis.

Susceptibility of Pseudomonas aeruginosa veterinary isolates to Pbunavirus PB1-like phages.

In recent years, antimicrobial-resistant Pseudomonas aeruginosa strains have increased in the veterinary field. Therefore, phage therapy has received significant attention as an approach for overcoming antimicrobial resistance. In this context, we isolated and characterized four Pseudomonas bacteriophages. Phylogenetic analysis showed that the isolated phages are novel Myoviridae Pbunavirus PB1-like phages with ØR12 belonging to a different clade compared with the other three. These phages had distinct lytic activity against 22 P. aeruginosa veterinary isolates. The phage cocktail composed from the PB1-like phages clearly inhibited the occurrence of the phage-resistant variant, suggesting that these phages could be useful in phage therapy.

Identification of a novel mechanism of action of bovine IgG antibodies specific for Staphylococcus aureus.

Staphylococcus aureus is a major pathogen that causes subclinical mastitis associated with huge economic losses to the dairy industry. A few vaccines for bovine mastitis are available, and they are expected to induce the production of S. aureus-specific antibodies that prevent bacterial adherence to host cells or promote opsonization by phagocytes. However, the efficacy of such vaccines are still under debate; therefore, further research focusing on improving the current vaccines by seeking additional mechanisms of action is required to reduce economic losses due to mastitis in the dairy industry. Here, we generated S. aureus-specific bovine IgG antibodies (anti-S. aureus) that directly inhibited bacterial growth in vitro. Inhibition depended on specificity for anti-S. aureus, not the interaction between Protein A and the fragment crystallizable region of the IgG antibodies or bacterial agglutination. An in vitro culture study using S. aureus strain JE2 and its deletion mutant JE2ΔSrtA, which lacks the gene encoding sortase A, revealed that the effect of anti-S. aureus was sortase-A-independent. Sortase A is involved in the synthesis of cell-wall-associated proteins. Thus, other surface molecules, such as membrane proteins, cell surface polysaccharides, or both, may trigger the inhibition of bacterial growth by anti-S. aureus. Together, our findings contribute insights into developing new strategies to further improve the available mastitis vaccine by designing a novel antigen on the surface of S. aureus to induce inhibitory signals that prevent bacterial growth.

Phage Therapy Is Effective in a Mouse Model of Bacterial Equine Keratitis.

UNLABELLED: Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, and pseudomonads. Of these bacteria, Pseudomonas aeruginosa sometimes causes rapid corneal corruption and, in some cases, blindness. Antimicrobial resistance can make treatment very difficult. Therefore, new strategies to control bacterial infection are required. A bacteriophage (phage) is a virus that specifically infects and kills bacteria. Since phage often can lyse antibiotic-resistant bacteria because the killing mechanism is different, we examined the use of phage to treat horse bacterial keratitis. We isolated Myoviridae or Podoviridae phages, which together have a broad host range. They adsorb efficiently to host bacteria; more than 80% of the ΦR18 phage were adsorbed to host cells after 30 s. In our keratitis mouse model, the administration of phage within 3 h also could kill bacteria and suppress keratitis. A phage multiplicity of infection of 100 times the host bacterial number could kill host bacteria effectively. A cocktail of two phages suppressed bacteria in the keratitis model mouse. These data demonstrated that the phages in this study could completely prevent the keratitis caused by P. aeruginosa in a keratitis mouse model. Furthermore, these results suggest that phage may be a more effective prophylaxis for horse keratitis than the current preventive use of antibiotics. Such treatment may reduce the use of antibiotics and therefore antibiotic resistance. Further studies are required to assess phage therapy as a candidate for treatment of horse keratitis. IMPORTANCE: Antibiotic-resistant bacteria are emerging all over the world. Bacteriophages have great potential for resolution of this problem. A bacteriophage, or phage, is a virus that infects bacteria specifically. As a novel therapeutic strategy against racehorse keratitis caused by Pseudomonas aeruginosa, we propose the application of phages for treatment. Phages isolated in this work had in vitro effectiveness for a broad range of P. aeruginosa strains. Indeed, a great reduction of bacterial proliferation was shown in phage therapy for mouse models of P. aeruginosa keratitis. Therefore, to reduce antibiotic usage, phage therapy should be investigated and developed further.

Effect of dietary eugenol on xenobiotic metabolism and mediation of UDP-glucuronosyltransferase and cytochrome P450 1A1 expression in rat liver.

Xenobiotic-metabolizing enzymes (XMEs) play an important role in the elimination and detoxification of xenobiotics and drugs. A variety of natural dietary agents are known to protect against cancer by inducing XME. To elucidate the molecular mechanism of XME induction, we examined the effect of dietary eugenol (4-allyl-1-hydroxy-2-methoxybenzene) on xenobiotic metabolism. In this study, rats were administered dietary eugenol for 4 weeks to investigate the various effects of UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) expression. In rats administered dietary eugenol, expression levels of hepatic CYP1A 1 were reduced to 40% than of the controls, while expression of hepatic UGT1A6, UGT1A7 and UGT2B1 increased to 2-3 times than observed in the controls. Hepatic protein levels of UGT1A6 and 2B1 were also elevated in the eugenol-treated rats. These results suggest that the natural compound eugenol improves the xenobiotic-metabolizing systems that suppress and induce the expression of CYP1A1 and UGT, respectively.

Using phage to drive selections toward restoring antibiotic sensitivity in Pseudomonas aeruginosa via chromosomal deletions.

Phage therapy has re-emerged in modern medicine as a robust antimicrobial strategy in response to the increasing prevalence of antimicrobial-resistant bacteria. However, bacterial resistance to phages can also arise via a variety of molecular mechanisms. In fact, several clinical studies on phage therapy have reported the occurrence of phage-resistant variants, representing a significant concern for the successful development of phage-based therapies. In this context, the fitness trade-offs between phage and antibiotic resistance have revealed new avenues in the field of phage therapy as a countermeasure against phage resistance. This strategy forces to restore the antibiotic susceptibility of antimicrobial-resistant bacteria as compensation for the development of phage resistance. Here, we present the key achievements of these fitness trade-offs, notably focusing on the enhancement of antibiotic sensitivity through the induction of large chromosomal deletions by bacteriophage infection. We also describe the challenges of this strategy that need to be overcome to promote favorable therapeutic outcomes and discuss future directions. The insights gained from the trade-offs between phage and antibiotic sensitivity will help maximize the potential of phage therapy for the treatment of infectious diseases.

DNA methylation of Ad4BP/SF-1 suppresses Cyp11a1 and StAR transcripts in C2C12 myoblasts.

Steroidogenesis occurs locally in peripheral tissues and via adrenal and gonadal glands' biosynthesis. The C2C12 mouse myoblast cell line and rat skeletal muscles harbor a local steroidogenesis pathway for glucocorticoids, and corticosterone is biosynthesized from skeletal muscle cells. However, Cyp11a1 and StAR protein expressions are not observed in C2C12 cells or rat muscular tissues. In this context, this study investigated the relationship between DNA methylation and key steroidogenic genes. Bioinformatics analysis of methylated DNA immune precipitation showed that C2C12 myoblasts and myotubes did not have remarkable DNA methylated regions in the gene-body of Cyp11a1. However, a highly methylated region in the CpG island was detected in the intronic enhancer of Ad4BP/SF-1, known as the transcriptional factor for steroidogenic genes. After C2C12 myoblasts treatment with 5-aza-2-deoxycytidine, the gene expressions of Ad4BP/SF-1, Cyp11a1, and StAR were significantly time- and concentration-dependent upregulated. To clarify the contribution of Ad4BP/SF-1 on Cyp11a1 and StAR transcripts, we silenced Ad4BP/SF-1 during the 5-aza-2-deoxycytidine treatment in C2C12 myoblasts, resulting in significant suppression of both Cyp11a1 and StAR. Additionally, pregnenolone levels in the supernatants of C2C12 cells were enhanced by 5-aza-2-deoxycytidine treatment, whereas pregnenolone production by C2C12 myoblasts was significantly suppressed by Ad4BP/SF-1 knockdown. These results indicate that DNA methylation of Ad4BP/SF-1 might be involved in the downregulation of steroidogenic genes, such as Cyp11a1 and StAR in C2C12 myoblasts.

Innate immune response of bovine mammary epithelial cells in Mycoplasma bovis mastitis using an in vitro model of bovine mammary gland infection.

Mycoplasma bovis mastitisis highly contagious and disrupts lactation, posing a significant threat to the dairy industry. While the mammary gland's defence mechanism involves epithelial cells and mononuclear cells (MNC), their interaction with M. bovis remains incompletely understood. In this study, we assessed the immunological reactivity of bovine mammary epithelial cells (bMEC) to M. bovis through co-culture with MNC. Upon co-culture with MNC, the mRNA expression levels of interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor (TNF)-α in bMEC stimulated by M. bovis showed a significant increase compared to monoculture. Additionally, when stimulated with M. bovis, the culture supernatant exhibited significantly higher concentrations of IL-6 and interferon (IFN)-γ, while IL-1ß concentration tended to be higher in co-culture with MNC than in monoculture. Furthermore, the mRNA expression levels of toll-like receptor (TLR) 2 in bMEC stimulated with M. bovis tended to increase, and TLR4 significantly increased when co-cultured with MNC compared to monocultures. However, the surface expression levels in bMEC did not exhibit significant changes between co-culture and monoculture. Overall, our research indicates that the inflammatory response of bMEC is increased during co-culture with MNC, suggesting that the interaction between bMEC and MNC in the mammary gland amplifies the immune response to M. bovis in cows affected by M. bovis mastitis.

Effects of the combination of anti-PcrV antibody and bacteriophage therapy in a mouse model of Pseudomonas aeruginosa pneumonia.

Acute lung injury caused by Pseudomonas aeruginosa is attributed to the translocation of cytotoxin into pulmonary epithelial cells via the P. aeruginosa type III secretion system. This virulence can be blocked with a specific antibody against PcrV in this secretion system. However, because anti-PcrV antibodies do not have bactericidal activity, the treatment of bacteria depends on the phagocytic system of the host. In this study, we investigated the therapeutic effect of combination therapy with an anti-PcrV antibody and bactericidal bacteriophages on acute lung injury and subsequent death in mice compared with a single treatment. After the mice intratracheally received a lethal dose of the cytotoxic P. aeruginosa strain, a second instillation was performed with saline, anti-PcrV IgG, bacteriophages, or a mixture of anti-PcrV and bacteriophages. The survival rates 24 h after infection were as follows: 7.1% in the saline group, 26.7% in the anti-PcrV group, 41.2% in the phage group, and 66.7% in the anti-PcrV + phage group (P < 0.001 vs saline-treated group). The activity of surviving mice in the anti-PcrV + phage group was significantly greater than that in the saline group. The lung weight in the anti-PcrV + phage group was significantly lower than that in the anti-PcrV group. In conclusion, combination therapy with an anti-PcrV antibody and a bacteriophage reduces acute lung injury and suggests improved survival compared with each treatment alone. This combination therapy, which does not rely on conventional antibiotics, could constitute a new strategy for treating multidrug-resistant P. aeruginosa infections.IMPORTANCECombination therapy with either bacteriophages alone or in combination with anti-PcrV antibodies in a mouse model of Pseudomonas aeruginosa pneumonia may reduce the acute lung injury and improve survival. This combination therapy, which does not rely on conventional antibiotics, may be a new strategy to treat multidrug-resistant Pseudomonas aeruginosa infections.

A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): a pilot study in a canine model.

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P<0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [(3)H]l-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P<0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM.

Mechanism of skeletal muscle atrophy by muscle fiber types in male rats under long-term fasting stress.

Fasting induces metabolic changes in muscles, which are differentiated by muscle fiber type. In this study, the mechanism of fasting-induced muscle atrophy in rats was examined to determine the differences between muscle fiber types in energy production. Fasting for 96 h did not alter the weight of the soleus (SOL), a fiber type I muscle, but did significantly reduce the weight of gastrocnemius (GM), a fiber type II muscle. GM, SOL and blood pregnenolone and testosterone levels decreased under fasting, which induced energy deprivation, whereas corticosterone (CORT) levels significantly increased. However, the expression of 3ß-HSD and P45011ß in GM was unaffected by fasting. The decrease in GM weight may be due to decreased levels of testosterone and reduced synthesis of mammalian target of rapamycin (mTOR). Significant increases in CORT both GM and SOL were associated with increases in the amount of branched-chain amino acids available for energy production. However, decreased levels of mTOR and IGF1 and increased levels of CORT and IL-6 in SOL suggest that GM proteolysis was followed by SOL proteolysis for additional energy production. In conclusion, IGF1 levels decreased significantly in SOL, whereas those of IL-6 significantly increased in SOL and blood but decreased in GM. Blood branched-chain amino acids (BCAA) levels were unaffected due to fasting, whereas an increase was noted in the levels of BCAA in GM and SOL. These results show that fasting for 96 h restricts energy supply, producing fast-twitch muscle atrophy followed by slow-twitch muscle atrophy.

16S rRNA nanopore sequencing for rapid diagnosis of causative bacteria in bovine mastitis.

The rapid identification of specific bacterial pathogens in bovine mastitis is crucial for appropriate antimicrobial treatment. Sequencing of 16S rRNA gene amplicons is a proven, useful strategy for diagnosing bacterial infections. In this study, the use of 16S rRNA analysis with nanopore sequencer for the rapid identification of causative bacteria in bovine mastitis, was evaluated. DNA was extracted from 122 milk samples from cattle with suspected mastitis based on clinical symptoms. 16S rRNA gene amplicon sequencing was conducted using a nanopore sequencer. The efficacy of bacterial identification was verified by comparison with conventional culture methods. Nanopore sequencing identified the causative bacteria with high accuracy within approximately 6 h from the time of sample collection. When the major causative bacteria of bovine mastitis (Escherichia coli, Streptcoccus uberis, Klebsiella pneumoniae, and Staphylococcus aureus) were detected by nanopore sequencing, 98.3% of the results were consistent with identification through conventional culturing methods. 16S rRNA gene analysis using a nanopore sequencer enabled the rapid and accurate identification of bacterial species in bovine mastitis.

Isolation of Streptococcus mutans temperate bacteriophage with broad killing activity to S. mutans clinical isolates.

Bacteriophages are expected to be therapeutic agents against infectious diseases. Streptococcus mutans are involved in dental plaque formation related to dental caries and periodontitis. In S. mutans, lytic bacteriophages have been isolated previously, but the isolation of temperate bacteriophage has not been reported although their presence in the genome has been confirmed. Here, we report the isolation of temperate bacteriophage, φKSM96, from S. mutans. φKSM96 has a circular DNA 39,820 bp long and reveals Siphoviridae morphology. φKSM96 shows a broad range of susceptibility against S. mutans strains with different serotypes. By the addition of φKSM96, S. mutans growth and biofilm formation were significantly inhibited. In cocultures of S. mutans with other bacterial species, the proportion of S. mutans significantly decreased in the presence of φKSM96. In summary, φKSM96 shows selective anti-S. mutans activity. The isolation of temperate bacteriophage is important for future genetic manipulation to create more efficient bacteriophages.

Antimicrobial susceptibility of bovine clinical mastitis pathogens in Japan and development of a simplified agar disk diffusion method for clinical practice.

This study aimed to examine the antimicrobial susceptibility of bovine mastitis pathogens in Japan and develop criteria for testing antimicrobial susceptibility using the simplified agar disk diffusion (ADD) method that is currently being used in clinical practice. Milk samples from 1,349 dairy cows with clinical mastitis were collected and cultured. The minimum inhibitory concentrations (MICs) of the antimicrobials were determined for 504 strains of 28 bacteria. Of the gram-positive bacteria, most Staphylococcus spp. were susceptible to penicillin G (PCG), kanamycin (KM), oxytetracycline (OTC), cefazolin (CEZ), pirlimycin, enrofloxacin, and marbofloxacin. Streptococcus spp. and Trueperella pyogenes showed resistance to OTC and KM. Most gram-negative bacteria were resistant to OTC and CEZ and particularly susceptible to fluoroquinolones. To develop the criteria for a disk diffusion test of the simplified ADD method, the relationships between MICs and diameters of inhibition zones (DIZs) were analyzed and compared with the conventional method. The susceptibility breakpoints of several antimicrobials were lower for both gram-positive and gram-negative bacteria. Particularly for gram-positive bacteria, the application of the new criteria lowers the breakpoint for PCG, suggesting that the use of PCG instead of CEZ may increase. The results suggest that use of these criteria for the simplified ADD method may lead to appropriate antimicrobial choice and consequently the appropriate use of antimicrobials in clinical practice.

Influence of the Hypoxia-Activated Prodrug Evofosfamide (TH-302) on Glycolytic Metabolism of Canine Glioma: A Potential Improvement in Cancer Metabolism.

The transcription factor hypoxia-inducible factor 1α (HIF-1α) drives metabolic reprogramming in gliomas (GLs) under hypoxic conditions, promoting glycolysis for tumor development. Evofosfamide (EVO) releases a DNA-alkylating agent within hypoxic regions, indicating that it may serve as a hypoxia-targeted therapy. The aim of this study was to investigate the glycolytic metabolism and antitumor effects of EVO in a canine GL model. Our clinical data showed that overall survival was significantly decreased in GL dog patients with higher HIF-1α expression compared to that of those with lower HIF-1α expression, and there was a positive correlation between HIF-1α and pyruvate dehydrogenase kinase 1 (PDK1) expression, suggesting that glycolytic activity under hypoxia conditions may contribute to poor outcomes in canine GL. Our glycolysis assay tests showed that the glycolytic ATP level was higher than the mitochondrial ATP level in three types of canine GL cell lines by activating the HIF-1 signal pathway under hypoxia conditions, resulting in an overall increase in total cellular ATP production. However, treatment with EVO inhibited the glycolytic ATP level in the GL cell lines under hypoxia conditions by targeting HIF-1α-positive cells, leading to decrease in total cellular ATP production. Our in vivo tests showed that EVO significantly reduced tumor development compared to controls and temozolomide in murine GL models. A metabolic analysis demonstrated that EVO effectively suppressed glycolytic metabolism by eliminating HIF-1α-positive cells, suggesting that it may restore metabolism in canine GLs. The evidence presented here supports the favorable preclinical evaluation of EVO as a potential improvement in cancer metabolism.

Histone H1 null vertebrate cells exhibit altered nucleosome architecture.

In eukaryotic nuclei, DNA is wrapped around an octamer of core histones to form nucleosomes, and chromatin fibers are thought to be stabilized by linker histones of the H1 type. Higher eukaryotes express multiple variants of histone H1; chickens possess six H1 variants. Here, we generated and analyzed the phenotype of a complete deletion of histone H1 genes in chicken cells. The H1-null cells showed decreased global nucleosome spacing, expanded nuclear volumes, and increased chromosome aberration rates, although proper mitotic chromatin structure appeared to be maintained. Expression array analysis revealed that the transcription of multiple genes was affected and was mostly downregulated in histone H1-deficient cells. This report describes the first histone H1 complete knockout cells in vertebrates and suggests that linker histone H1, while not required for mitotic chromatin condensation, plays important roles in nucleosome spacing and interphase chromatin compaction and acts as a global transcription regulator.

Relationship between Escherichia coli virulence factors, notably kpsMTII, and symptoms of clinical metritis and endometritis in dairy cows.

Although Escherichia coli is a commensal bacterium of the bovine vaginal microbiota, it is an important pathogenic bacterium that causes diseases of the reproductive tract and sub-fertility. Recent studies have focused on virulence factors (VFs) of intrauterine E. coli; however, actual endometrial VFs have not been clearly identified. The purpose of this study was to identify the VFs of E. coli associated with clinical metritis and endometritis. Thirty-two strains of E. coli and four mixed Trueperella pyogenes (TP) strains were detected in the uterus of 19 Holstein dairy cows with obvious clinical signs (between 8 and 66 days postpartum). The presence of six E. coli VFs (fimH, fyuA, kpsMTII, hra1, csgA, and astA) was examined by PCR, and clinical signs and reproductive performance (mixed TP, the percentage of polymorphonuclear neutrophils [PMN%], days to uterine involution, etc.) were evaluated. Four VFs (fimH, hra1, csgA, and astA) were detected in all E. coli strains, whereas fyuA and kpsMTII were detected in 94% and 50% of strains, respectively. Cows with E. coli strains harboring kpsMTII exhibited significantly severe clinical scores (vaginal discharge score, PMN%, uterine involution), suggesting that kpsMTII is a key VF for progression of clinical metritis and endometritis. In the present study, we clearly identified six VFs associated with clinical metritis and endometritis. In addition, E. coli strains with kpsMTII probably play a crucial role in the progression of clinical metritis and endometritis.

Bisphenol A-sulfate conjugate disrupts AURKA transcription and cell cycle in BeWo cytotrophoblasts.

Bisphenol A (BPA) has been shown to exhibit various toxic effects, including the induction of reproductive disorders. Generally, BPA is converted to conjugated metabolites, leading to bio-inactivation. On the other hand, the toxicity of conjugated metabolites is not fully understood. Notably, the placenta develops the sulfate-sulfatase pathway, which transports and reactivates sulfated steroids. Therefore, we investigated the potential adverse effects of the BPA-sulfate conjugate (BPA-S) on human placenta-derived BeWo cytotrophoblasts. In the present study, high-concentration BPA-S (100 µM) induced significant inhibition of BeWo growth, with effects similar to those seen with unconjugated BPA (100 µM and 100 nM). This growth inhibition was restored by treatment of the cells with an inhibitor of the organic anion-transporting peptides (OATPs) (bromosulphophthalein) or with a sulfatase (STS) inhibitor (STX64). BeWo exhibits expression of the genes encoding OATP1A2 and OATP4A1 as known sulfated steroid transporters and STS, suggesting that BPA-S suppresses cell growth activity via the sulfate-sulfatase pathway. In addition, cell cycle analysis revealed that BPA-S (100 µM) increased the fraction of cytotrophoblasts in the G2/M phases and significantly decreased the accumulation of the transcript encoding Aurora kinase A (AURKA), which is a critical regulator of cellular division. These results suggested that BPA-S triggers cell cycle arrest and inhibits proliferation of BeWo cytotrophoblasts by decreased AURKA, an effect that is mediated by the sulfate-sulfatase pathway. Overall, these findings provide insights into the reactivation of sulfated endocrine-disrupting chemicals and subsequent adverse effects.