Transcriptional Profiling of the Effect of Coleus amboinicus L. Essential Oil against Salmonella Typhimurium Biofilm Formation.

Salmonella enterica serovar Typhimurium cause infections primarily through foodborne transmission and remains a significant public health concern. The biofilm formation of this bacteria also contributes to their multidrug-resistant nature. Essential oils from medicinal plants are considered potential alternatives to conventional antibiotics. Therefore, this study assessed the antimicrobial and antibiofilm activities of Coleus amboinicus essential oil (EO-CA) against S. Typhimurium ATCC 14028. Seventeen chemical compounds of EO-CA were identified, and carvacrol (38.26%) was found to be the main constituent. The minimum inhibitory concentration (MIC) of EO-CA for S. Typhimurium planktonic growth was 1024 µg/mL while the minimum bactericidal concentration was 1024 µg/mL. EO-CA at sub-MIC (≥1/16× MIC) exhibited antibiofilm activity against the prebiofilm formation of S. Typhimurium at 24 h. Furthermore, EO-CA (≥1/4× MIC) inhibited postbiofilm formation at 24 and 48 h (p < 0.05). Transcriptional profiling revealed that the EO-CA-treated group at 1/2× MIC had 375 differentially expressed genes (DEGs), 106 of which were upregulated and 269 were downregulated. Five significantly downregulated virulent DEGs responsible for motility (flhD, fljB, and fimD), curli fimbriae (csgD), and invasion (hilA) were screened via quantitative reverse transcription PCR (qRT-PCR). This study suggests the potential of EO-CA as an effective antimicrobial agent for combating planktonic and biofilm formation of Salmonella.

Effects of Piper betle Extracts against Biofilm Formation by Methicillin-Resistant Staphylococcus pseudintermedius Isolated from Dogs.

Emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolated from dogs with cutaneous and wound infections has significantly impacted veterinary medicine. This study aimed to isolate S. pseudintermedius from canine pyoderma and investigate the effects of ethanolic extracts of Piper betle (PB), P. sarmentosum (PS), and P. nigrum (PN) on the bacterial growth and biofilm formation of S. pseudintermedius and MRSP. Of the isolated 152 isolates, 53 were identified as S. pseudintermedius using polymerase chain reaction, and 10 isolates (6.58%) were identified as MRSP based on the presence of mecA. Based on phenotype, 90% of MRSPs were multidrug-resistant. All MRSP had moderate (10%, 1/10) and strong (90%, 9/10) biofilm production ability. PB extracts were the most effective in inhibiting planktonic cells, and the minimum inhibitory concentration at which ≥50% of the isolates were inhibited (MIC50) was 256 µg/mL (256-1024 µg/mL) for S. pseudintermedius isolates and 512 µg/mL (256-1024 µg/mL) for MRSP isolates. The MIC90 for S. pseudintermedius and MRSP was 512 µg/mL. In XTT assay, PB at 4× MIC showed an inhibition rate of 39.66-68.90% and 45.58-59.13% for S. pseudintermedius and MRSP, respectively, in inhibiting biofilm formation. For PB at 8× MIC, the inhibition rates for S. pseudintermedius and MRSP were 50.74-81.66% and 59.57-78.33%, respectively. Further, 18 compounds were identified in PB using gas chromatography-mass spectrometry, and hydroxychavicol (36.02%) was the major constituent. These results indicated that PB could inhibit bacteria growth of and biofilm formation by S. pseudintermedius and MRSP isolated from canine pyoderma in a concentration-dependent manner. Therefore, PB is a potential candidate for the treatment of MRSP infection and biofilm formation in veterinary medicine.

Effects of Piper nigrum L. Fruit Essential Oil Toxicity against Stable Fly (Diptera: Muscidae).

The efficacy of Piper nigrum L. fruit essential oil (EO) against Stomoxys calcitrans (stable fly), a blood-feeding fly distributed worldwide, was investigated. This study aimed to evaluate the insecticidal activity of EO based on contact and fumigant toxicity tests. Chemical analysis of the EO using gas chromatography-mass spectrometry revealed that sabinene (24.41%), limonene (23.80%), ß-caryophyllene (18.52%), and α-pinene (10.59%) were the major components. The results demonstrated that fly mortality increased with increasing EO concentration and time during the first 24 h of exposure. The median lethal dose was 78.37 µg/fly for contact toxicity, while the 90% lethal dose was 556.28 µg/fly. The median lethal concentration during fumigant toxicity testing was 13.72 mg/L air, and the 90% lethal concentration was 45.63 mg/L air. Our findings suggested that essential oil extracted from P. nigrum fruit could be a potential natural insecticidal agent for control of stable fly. To examine the insecticidal properties of P. nigrum fruit EO, further field trials and investigation into the efficacy of nano-formulations are required.

Effects of Coleus amboinicus L. Essential Oil and Ethanolic Extracts on Planktonic Cells and Biofilm Formation of Microsporum canis Isolated from Feline Dermatophytosis.

Microsporum canis is an important zoonotic fungus that causes dermatophytosis in domestic animals and their owners. Domestic cats are the primary reservoir for M. canis. Antifungal drugs frequently produce adverse effects on the host animal, increasing the demand for novel alternative treatments derived from nature. We evaluated the antifungal activity of Coleus amboinicus essential oil (CEO) and ethanolic extracts (CEE) against M. canis in planktonic and biofilm growth. Twelve clinical isolates of M. canis were identified in feline dermatophyte samples. Using GC-MS, 18 compounds were identified in CEO, with carvacrol being the major constituent. HPLC analysis of CEE revealed that it contained rosmarinic acid, apigenin, and caffeic acid. The planktonic growth of all M. canis isolates was inhibited by C. amboinicus extracts. The minimum inhibitory concentration at which ≥50% of the isolates were inhibited (MIC50) was 128 µg/mL (32-256 µg/mL) for both CEO and CEE. The MIC90 values of CEO and CEE were 128 and 256 µg/mL, respectively. CEO at MIC (128 µg/mL) and 2× MIC (256 µg/mL) significantly inhibited the biofilm formation of weak, moderate, and strong biofilm-producing M. canis. CEE at 2× MIC (256 µg/mL) significantly inhibited the biofilm formation of all isolates. Overall, C. amboinicus extracts inhibited planktonic growth and exhibited a significant antibiofilm effect against M. canis. Thus, C. amboinicus is a potential source of natural antifungal compounds.

Natural Antioxidant, Antibacterial, and Antiproliferative Activities of Ethanolic Extracts from Punica granatum L. Tree Barks Mediated by Extracellular Signal-Regulated Kinase.

The nonedible parts of the pomegranate plant, such as tree barks and fruit peels, have pharmacological properties that are useful in traditional medicine. To increase their value, this study aimed to compare the antioxidative and antibacterial effects of ethanolic extracts from pomegranate barks (PBE) and peels (PPE). The antiproliferative effects on HeLa and HepG2 cells through the extracellular signal-regulated kinase pathway were also evaluated. The results indicated that the total amounts of phenolics and flavonoids of PBE and PPE were 574.64 and 242.60 mg equivalent gallic acid/g sample and 52.98 and 23.08 mg equivalent quercetin/g sample, respectively. Gas chromatography−mass spectrometry revealed that 5-hdroxymethylfurfural was the major component of both PBE (23.76%) and PPE (33.19%). The 2,2-diphenyl-1-picryl-hydrazyl-hydrate free radical scavenging capacities of PBE and PPE, in terms of the IC50 value, were 4.1 and 9.6 µg/mL, respectively. PBE had a greater potent antibacterial effect against Escherichia coli, Staphylococcus aureus, Salmonella Enteritidis, and S. Typhimurium. PBE and PPE (1000 µg/mL) had exhibited no cytotoxic effects on LLC-MK2. PBE and PPE (250 and 1000 µg/mL, respectively) treatments were safe for BHK-21. Both extracts significantly inhibited HepG2 and HeLa cell proliferations at 10 and 50 µg/mL, respectively (p < 0.001). The results indicated that PBE and PPE have remarkable efficiencies as free radical scavengers and antibacterial agents, with PBE exhibiting greater efficiency. The inhibitory effects on HepG2 might be through the modulation of the ERK1/2 expression. PBE and PPE have the potential for use as optional supplementary antioxidative, antibacterial, and anticancer agents.

Contact and Fumigant Activities of Citrus aurantium Essential Oil against the Stable Fly Stomoxys calcitrans (Diptera: Muscidae).

The stable fly, Stomoxys calcitrans (L.), is a cosmopolitan hematophagous fly of medical and veterinary importance. It is widely considered a major livestock pest that can cause significant economic losses. This study aimed to evaluate the insecticidal activity of Citrus aurantium (L.) essential oil against S. calcitrans based on contact and fumigant toxicity tests. Chemical analysis by gas chromatography-mass spectrometry of the essential oil showed the dominance (93.79%) of limonene in the total essential oil composition. Furthermore, the insecticidal test results showed that the mortality of flies increased with concentration and time within 24 h of exposure. In the contact toxicity test, the median lethal dose was 105.88 µg/fly, while the 90% lethal dose was 499.25 µg/fly. As for the fumigant toxicity test, the median lethal concentration was 13.06 mg/L air, and the 90% lethal concentration was 43.13 mg/L air. These results indicate that C. aurantium essential oil exhibits insecticidal activity against S. calcitrans. Therefore, it can be used as an alternative to synthetic insecticides for achieving stable fly control.

Insecticidal Activity of Plectranthus amboinicus Essential Oil against the Stable Fly Stomoxys calcitrans (Diptera: Muscidae) and the Horse Fly Tabanus megalops (Diptera: Tabanidae).

The stable fly, Stomoxys calcitrans (Diptera: Muscidae), and the horse fly, Tabanus megalops (Diptera: Tabanidae), are important ectoparasites of livestock in Thailand. These species affect animal health and cause economic losses. This study investigated the insecticidal activity of Plectranthus amboinicus essential oil against S. calcitrans and T. megalops through contact and fumigant toxicity tests and evaluated the effects of the essential oil on these flies through histopathological and scanning electron microscopic (SEM) studies. The results of the contact toxicity test indicated that the median lethal dose against S. calcitrans and T. megalops was 12.05 and 131.41 µg/fly, and the 90% lethal dose was 45.53 and 200.62 µg/fly, respectively. The results of the fumigant toxicity test showed that the median lethal concentration against S. calcitrans and T. megalops was 1.34 and 7.12 mg/L air, and the 90% lethal concentration was 4.39 and 30.37 mg/L air, respectively. Histopathology revealed neuronal degeneration in the brain of S. calcitrans and interstitial neuronal edema of the brain and ovarian necrosis in T. megalops. No external morphological changes were observed via SEM. Given its insecticidal properties against S. calcitrans and T. megalops, P. amboinicus essential oil could be developed into a natural insecticide to control these fly species.

Polyether ionophore kijimicin inhibits growth of Toxoplasma gondii and controls acute toxoplasmosis in mice.

The natural polyether ionophore antibiotics may be important chemotherapeutic agents. Among them, kijimicin represents an important type of ionophore compound because it inhibits Eimeria tenella and human immunodeficiency virus. The ionophore monensin displays potent activities against several coccidian parasites including the opportunistic pathogen of humans, Toxoplasma gondii. At first, we evaluated the anti-Toxoplasma activity of kijimicin, monensin as a reference control, and anti-Toxoplasma drugs such as clindamycin, in vitro. The half inhibitory concentrations (IC50) for the anti-Toxoplasma activities of kijimicin, monensin, and clindamycin were 45.6 ± 2.4 nM, 1.3 ± 1.8 nM, and 238.5 ± 1.8 nM, respectively. Morphological analyses by electron microscopy revealed cellular swelling and multiple intracellular vacuole-like structures in the T. gondii tachyzoites after treatment with kijimicin and monensin. Kijimicin and monensin also inhibited the invasion of extracellular parasites (IC50 = 216.6 ± 1.9 pM and 531.1 ± 1.9 pM, respectively). Importantly, kijimicin treatment resulted in decreased mitochondrial membrane potential and generation of reactive oxygen species in T. gondii as monensin did. Furthermore, mice treated with kijimicin at 10 mg/kg/day and 3 mg/kg/day showed 91.7% and 66.7% survival rates, respectively, 30 days after infection with T. gondii. The control mice all died within 18 days of infection. The present study shows that kijimicin inhibits T. gondii growth and changes the ultrastruct of the parasites. This finding may lead to validation of kijimicin as new drug to control T. gondii growth.

Metacytofilin has potent anti-malarial activity.

Metacytofilin (MCF) was isolated from the fungus Metarhizium sp. TA2759. Although MCF possesses anti-Toxoplasma activity, the effects of this compound against other parasites are unknown. Here, we evaluated the in vitro anti-malarial activity of MCF against the 3D7 strain and the chloroquine-resistant K1 strain of Plasmodium falciparum. The half maximal inhibitory concentrations (IC50) of MCF against the 3D7 and K-1 strains following culture for 48 h were 666 nM and 605 nM, respectively. Artemisinin was more potent than MCF against both strains (3D7 IC50: 17.4 nM; K-1 IC50: 18.3 nM), while chloroquine was ineffective against the chloroquine-resistant strain (3D7 IC50: 39.1 nM; K-1 IC50: 1.62 µM). MCF affected the ring stage of the parasites, resulting in their death as shown by spots within red blood cells. MCF also inhibited parasite growth following culture for 72 h (3D7 IC50, 285 nM). Four optical isomers of cyclo[Leu-Phe]-diketopiperazine derivatives with modified methoxy and/or hydroxyl groups lost anti-malarial activity, suggesting that the spatial positions of the methoxy and hydroxyl groups in MCF play an important role in its anti-malarial effects. Together, these data suggest that MCF may represent a promising lead compound for treatment of drug-resistant malarial parasites.

Metacytofilin Is a Potent Therapeutic Drug Candidate for Toxoplasmosis.

BACKGROUND: Toxoplasmosis, a parasitic disease caused by Toxoplasma gondii, is an important cause of miscarriage or adverse fetal effects, including neurological and ocular manifestations in humans. Current anti-Toxoplasma drugs have limited efficacy against toxoplasmosis and also have severe side effects. Therefore, novel efficacious drugs are urgently needed. Here, we identified metacytofilin (MCF) from a fungal Metarhizium species as a potential anti-Toxoplasma compound. METHODS: Anti-Toxoplasma activities of MCF and its derivatives were evaluated in vitro and in vivo using nonpregnant and pregnant mice. To understand the mode of action of MCF, the RNA expression of host and parasite genes was investigated by RNAseq. RESULTS: In vitro, MCF inhibited the viability of intracellular and extracellular T. gondii. Administering MCF intraperitoneally or orally to mice after infection with T. gondii tachyzoites increased mouse survival compared with the untreated animals. Remarkably, oral administration of MCF to pregnant mice prevented vertical transmission of the parasite. Interestingly, RNA sequencing of T. gondii-infected cells treated with MCF showed that MCF inhibited DNA replication and enhanced RNA degradation in the parasites. CONCLUSIONS: With its potent anti-T. gondii activity, MCF is a strong candidate for future drug development against toxoplasmosis.

Ethanol Extracts from Thai Plants have Anti-Plasmodium and Anti-Toxoplasma Activities In Vitro.

PURPOSE: The ethanol extracts from seven Thai plants, Kaempferia parviflora, Stemona tuberosa Lour., Ananas comosus, Punica granatum, Musa sapientum L., Pseuderanthemum palatiferum and Annona muricata L., which are traditionally used in Thailand to support human health, were evaluated for their anti-Plasmodium and anti-Toxoplasma activities, and for their cytotoxicities against human foreskin fibroblasts in vitro. RESULTS: The K. parviflora, P. palatiferum and A. muricata extracts were active against P. falciparum (3D7) with selectivity index (SI) values > 10, while their half maximal inhibitory concentrations (IC50) were 28.7 µg/ml, 78.8 µg/ml and 46.1 µg/ml, respectively. Extracts from K. parviflora and M. sapientum (ripe fruit peel) inhibited T. gondii (RH) growth with IC50 values of 53.5 µg/ml and 90.4 µg/ml, respectively. The SI values of the extracts from K. parviflora and M. sapientum (ripe fruit peel) were 9.0 and 10.8, respectively. CONCLUSION: Our data show that some of the aforementioned ethanol extracts are potential sources of new drugs to treat Plasmodium and Toxoplasma infections.

Effects of Thai piperaceae plant extracts on Neospora caninum infection.

Neosporosis has a worldwide distribution and causes economic losses in farming, particularly by increasing the risk of abortion in cattle. This study investigated the effects of Thai piperaceae (Piper betle, P. nigrum, and P. sarmentosum) extracts on Neospora caninum infections in vitro and in vivo. In an in vitro parasite growth assay based on the green fluorescent protein (GFP) signal, P. betle was the most effective extract at inhibiting parasite growth in human foreskin fibroblast cells (IC50 of GFP-expressing N. caninum parasites, 22.1µg/ml). The P. betle extract, at 25µg per ml, inhibited parasite invasion into host cells. Furthermore, in two independent experiments, treating N. caninum-infected mice with the P. betle extract for 7days post-infection increased their survival. In trial one, the anti-N. caninum effects of the P. betle extract reduced the mouse clinical scores for 30days post-infection (dpi). The survival rate of the mice treated with 400mg/kg was 100% compared with 66.6% for those treated with 100mg/kg and the non-treated controls. In trial two, treating the infected mice with the P. betle extract increased their survival at 50dpi. All mice in the non-treatment group died; however, the survival rates of the 400mg/kg-treated and 100mg/kg-treated mice were 83.3% and 33.3%, respectively. Also, a trend towards a reduced parasite burden was noted in the brains of the P. betle extract-treated mice, compared with the control mice. Therefore P. betle extract has potential as a medicinal plant for treating neosporosis.

Effects of Extracts from Thai Piperaceae Plants against Infection with Toxoplasma gondii.

Herbal medicines and natural herb extracts are widely used as alternative treatments for various parasitic diseases, and such extracts may also have potential to decrease the side effects of the standard regimen drugs used to treat toxoplasmosis (sulfadiazine-pyrimethamine combination). We evaluated how effective the Thai piperaceae plants Piper betle, P. nigrum and P. sarmentosum are against Toxoplasma gondii infection in vitro and in vivo. Individually, we extracted the piperaceae plants with ethanol, passed them through a rotary evaporator and then lyophilized them to obtain crude extracts for each one. The in vitro study indicated that the P. betle extract was the most effective extract at inhibiting parasite growth in HFF cells (IC50 on RH-GFP: 23.2 µg/mL, IC50 on PLK-GFP: 21.4 µg/mL). Furthermore, treatment of experimental mice with the P. betle extract for 7 days after infection with 1,000 tachyzoites of the T. gondii PLK strain increased their survival (survival rates: 100% in 400 mg/kg-treated, 83.3% in 100 mg/kg-treated, 33.3% in 25 mg/kg-treated, 33.3% in untreated mice). Furthermore, treatment with 400 mg/kg of the P. betle extract resulted in 100% mouse survival following infection with 100,000 tachyzoites. The present study shows that P. betle extract has the potential to act as a medical plant for the treatment of toxoplasmosis.

In vitro and in vivo effects of the phytohormone inhibitor fluridone against Neospora caninum infection.

Neospora caninum causes abortion and stillbirth in cattle. Identification of effective drugs against this parasite remains a challenge. Previous studies have suggested that disruption of abscisic acid (ABA)-mediated signaling in apicomplexan parasites such as Toxoplasma gondii offers a new drug target. In this study, the ABA inhibitor, fluridone (FLU), was evaluated for its action against N. caninum. Production of endogenous ABA within N. caninum was confirmed by ultra-performance liquid chromatography-tandem quadruple mass spectrometry. Subsequently, FLU treatment efficacy was assessed using in vitro. Results revealed that FLU inhibited the growth of N. caninum and T. gondii in vitro (IC50 143.1±43.96µM and 330.6±52.38µM, respectively). However, FLU did not affect parasite replication at 24h post-infection, but inhibited egress of N. caninum thereafter. To evaluate the effect of FLU in vivo, N. caninum-infected mice were treated with FLU for 15days. FLU treatment appeared to ameliorate acute neosporosis induced by lethal parasite challenge. Together, our data shows that ABA might control egress in N. caninum. Therefore, FLU has potential as a candidate drug for the treatment of acute neosporosis.