Antibacterial, Antibiofilm, and Antioxidant Activities of Aqueous Crude Gymnema inodorum Leaf Extract against Vancomycin-Resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VREF) causes nosocomial infections with high mortality and morbidity rates. This study aimed to evaluate the antibacterial and antibiofilm activities of aqueous crude Gymnema inodorum leaf extract (GIE) against the VREF ATCC 700221 strain. The antimicrobial activity of GIE against VREF was performed using disk diffusion and broth microdilution. The antibiofilm activities were evaluated using the crystal violet staining assay. The antioxidant potential was evaluated. Preliminary screening of the antimicrobial activity of 50 and 100 µg/disk of GIE against VREF revealed inhibition zones of 8.33 ± 0.58 mm and 8.67 ± 0.29 mm, respectively. Additionally, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against VREF were 125 and ≥ 250 mg/mL, respectively. SEM analysis showed that treatment with GIE caused morphological changes, including incomplete cell division, damaged cell walls, and cell content leakage, suggesting a disruption of bacterial cells. GIE also inhibited and eradicated biofilms formed by VREF. The extract exhibited antioxidant activities in the DPPH and ABTS assays. While GIE shows potential as an antibacterial and antibiofilm agent, further studies are necessary to fully understand the underlying mechanisms and optimize its use for therapeutic applications.

Occurrence of multidrug-resistant Mycobacterium tuberculosis in upper Southern Thailand.

Background and Aim: Mycobacterium tuberculosis causes global concern with tuberculosis (TB). Multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) pose additional challenges, as they resist to multiple first-line drugs. This study investigated the occurrence of TB, antibiotic resistance due to inhA and katG gene mutations, and multidrug resistance in M. tuberculosis during fiscal years 2020-2022. Materials and Methods: Samples were gathered from hospitals in seven provinces of upper Southern Thailand. The study investigated the correlation between inhA and katG gene mutations in M. tuberculosis and the development of antimicrobial resistance and isoniazid resistance. Results: A total of 19,186 samples were sent to the Office of Disease Prevention and Control Region 11st, Nakhon Si Thammarat, Thailand. The results showed that 51% of the samples were obtained from patients located in Nakhon Si Thammarat, followed by Surat Thani provinces. Regarding the spatial distribution of TB-infected cases, the incidence of TB was high in the province, which has a moderate to high population density. The highest average occurrence of TB in this study was found in Phuket province (9.75/100,000 risk person-year). The detected isoniazid resistance was 394, 255, and 179 cases in 2020, 2021, and 2022, respectively. A total of 99 isolates were MDR, whereas four isolates were XDR. The antimicrobial resistance associated with the inhA mutation was 192, 142, and 105 isolates, respectively, whereas the resistance associated with the katG mutation was 249, 182, and 120 cases in 2020, 2021, and 2022, respectively. Conclusion: These findings contribute to the understanding of the occurrence of antibiotic-resistant TB that could lead to use as data for preventing MDR-TB.

Antibacterial and antibiofilm efficacy of Solanum lasiocarpum root extract synthesized silver/silver chloride nanoparticles against Staphylococcus haemolyticus associated with bovine mastitis.

Staphylococcus haemolyticus is a cause of bovine mastitis, leading to inflammation in the mammary gland. This bacterial infection adversely affects animal health, reducing milk quality and yield. Its emergence has been widely reported, representing a significant economic loss for dairy farms. Interestingly, S. haemolyticus exhibits higher levels of antimicrobial resistance than other coagulase-negative Staphylococci. In this study, we synthesized silver/silver chloride nanoparticles (Ag/AgCl-NPs) using Solanum lasiocarpum root extract and evaluated their antibacterial and antibiofilm activities against S. haemolyticus. The formation of the Ag/AgCl-NPs was confirmed using UV-visible spectroscopy, which revealed maximum absorption at 419 nm. X-ray diffraction (XRD) analysis demonstrated the crystalline nature of the Ag/AgCl-NPs, exhibiting a face-centered cubic lattice. Fourier transform infrared (FT-IR) spectroscopy elucidated the functional groups potentially involved in the Ag/AgCl-NPs synthesis. Transmission electron microscopy (TEM) analysis revealed that the average particle size of the Ag/AgCl-NPs was 10 nm. Antimicrobial activity results indicated that the minimum inhibitory concentration (MIC) and maximum bactericidal concentration (MBC) of the Ag/AgCl-NPs treatment were 7.82-15.63 µg/mL towards S. haemolyticus. Morphological changes in bacterial cells treated with the Ag/AgCl-NPs were observed under scanning electron microscopy (SEM). The Ag/AgCl-NPs reduced both the biomass of biofilm formation and preformed biofilm by approximately 20.24-94.66 % and 13.67-88.48 %. Bacterial viability within biofilm formation and preformed biofilm was reduced by approximately 21.56-77.54 % and 18.9-71.48 %, respectively. This study provides evidence of the potential of the synthesized Ag/AgCl-NPs as an antibacterial and antibiofilm agent against S. haemolyticus.

Antibiograms, multidrug resistance, and milk-related parameters of bacteria isolated from milk of dairy cattle in Phatthalung, Thailand.

Background and Aim: Milk, a nutritious food, is widely consumed in human diets; however, contamination by micro-organisms can negatively impact its quality and consumer health. Contamination by micro-organisms affects the quality of milk, which can affect the quality of the milk production chain. This study aimed to determine the changes in milk composition and antibiotic susceptibility related to bacteria isolated from dairy cow milk. Materials and Methods: Raw milk samples were collected from 72 dairy cows. All milk samples were subjected to the California Mastitis Test (CMT) for CMT score determination. We also investigated milk composition, bacterial culture (BC), and antibiotic susceptibility. Results: About 47.22% and 30.56% of dairy cattle were positive for CMT + BC and automatic somatic cell count (ASCC) + BC, respectively. Fecal appearance and animal age were found to be risk factors for ASCC + BC positivity in dairy cattle. Bacteria were found in approximately 76% of milk samples, with the most common isolated species being α-hemolytic Streptococcus spp., coagulase-negative Staphylococcus spp., and Escherichia coli. Of these, 70% are resistant to at least one antibiotic. Variation in the multidrug resistance pattern was high in Klebsiella spp. Conclusions: Fecal appearance and animal age are risk factors for ASCC + BC positivity in dairy cattle. This study identified antibiotic and multidrug resistance patterns, which require comprehensive studies and effective surveillance systems. Remarkably, the use of antibiotic therapy in dairy cattle should be monitored.

Inhibitory and anti-adherent effects of Piper betle L. leaf extract against Acanthamoeba triangularis in co-infection with Staphylococcus aureus and Pseudomonas aeruginosa: A sustainable one-health approach.

Background and Aim: Keratitis is a serious ocular infection often caused by pathogenic microorganisms such as Acanthamoeba spp. Among other harmful microbes, Acanthamoeba keratitis presents a particular challenge due to its resistance to conventional antimicrobial agents. Piper betle Linn., commonly known as betel leaf, has been traditionally used for its medicinal properties. This study aimed to assess the potential of the leaf ethanol extract of P. betle Linn. in the treatment of Acanthamoeba triangularis in monoculture and co-culture with two prevalent pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, associated with keratitis. Materials and Methods: Minimum inhibitory concentrations (MICs) of A. triangularis, S. aureus, and P. aeruginosa extracts in monoculture and coinfected conditions were examined. In addition, this study explored the potential of the extract in preventing Acanthamoeba adherence in both monoculture and co-culture environments. Scanning electron microscopy (SEM) analysis confirmed the impact of the extract on Acanthamoeba cell membranes, including acanthopodia. Furthermore, a time-kill kinetic assay was used to validate the amoebicidal activity of the extract against A. triangularis and the tested bacteria. Results: MICs for trophozoites, cysts, P. aeruginosa, and S. aureus in the monoculture were 0.25, 0.25, 0.51, and 0.128 mg/mL, respectively, whereas the MICs for Acanthamoeba coinfected with bacteria were higher than those in the monoculture. This extract inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. Moreover, P. betle extract effectively prevented the adherence of Acanthamoeba to contact lenses under monoculture conditions. SEM analysis confirmed that P. betle extract affects the cell membrane of Acanthamoeba, including Acanthopodia. In addition, the time-kill kinetic assay confirmed that the extract contained amoebicidal activity against A. triangularis, including the tested bacteria. Notably, S. aureus was more susceptible than A. triangularis and P. aeruginosa to P. betle extract treatment. Unexpectedly, our study revealed that S. aureus negatively affected A. triangularis in the co-culture after 3 days of incubation, whereas P. aeruginosa facilitated the growth of A. triangularis in the presence of the extract. Conclusion: This study provides compelling evidence of the anti-adhesive and anti-Acanthamoeba properties of P. betle leaf extract against A. triangularis under monoculture and co-culture conditions. The observed impact on Acanthamoeba cell membranes, coupled with the time-kill kinetic assay results, underscores the potential of P. betle leaf extract as a promising agent for combating Acanthamoeba-related infections in humans and animals.

The infection of Cysticercus fasciolaris in natural rats (Rattus species) residing in human residence areas, Nakhon Si Thammarat, Thailand.

Cysticercus fasciolaris (C. fasciolaris) is the larval stage of a cestode parasite named Taenia taeniaeformis (T. taeniaeformis). C. fasiolaris is found in small rodents, especially rats. Rattus species are listed as intermediate hosts of this parasite, and cats are the main definitive host of C. fasiolaris. The objective of this study was to study the pathological, microscopic, and molecular aspects of C. fasciolaris in rodents residing in human residence areas. One hundred and two rodents were trapped in human settlements and dissected for larva-containing cyst examinations in the body cavity. The larvae of C. fasciolaris were investigated using histopathological examination, microscopic observations under a stereomicroscope and scanning electron microscope, and molecular detection using polymerase chain reaction. The prevalence of hepatic cysts containing larvae was 8.91% (95% CI = 4.16-16.24). In addition, the older larvae also had longer micropapillae. Histopathological investigation revealed normal hepatic tissue containing larvae and a scanty fluid cyst. The cyst capsule contains mostly mononuclear cells and spindle cells in all infected rats. The molecular detection using two primer sets revealed the amplicons were similar to the clade of C. fasciolaris. In the future, more investigation is necessary to fully understand the parasite's molecular pathogenesis and virulent molecules, which are less obvious.

Preparation and evaluation of a niosomal delivery system containing G. mangostana extract and study of its anti-Acanthamoeba activity.

Garcinia mangostana extract (GME) has severe pharmacokinetic deficiencies and is made up of a variety of bioactive components. GME has proven its anti-Acanthamoeba effectiveness. In this investigation, a GME-loaded niosome was developed to increase its potential therapeutic efficacy. A GME-loaded niosome was prepared by encapsulation in a mixture of span60, cholesterol, and chloroform by the thin film hydration method. The vesicle size, zeta potential, percentage of entrapment efficiency, and stability of GME-loaded niosomes were investigated. The values for GME-loaded niosome size and zeta potential were 404.23 ± 4.59 and -32.03 ± 0.95, respectively. The delivery system enhanced the anti-Acanthamoeba activity, which possessed MIC values of 0.25-4 mg mL-1. In addition, the niosomal formulation decreased the toxicity of GME by 16 times. GME-loaded niosome must be stored at 4 °C, as the quantity of remaining GME encapsulated is greater at this temperature than at room temperature. SEM revealed the damage to the cell membrane caused by trophozoites and cysts, which led to dead cells. In light of the above, it was found that GME-loaded niosomes had better anti-Acanthamoeba activity. The study suggested that GME-loaded niosomes could be used as an alternative to Acanthamoeba's therapeutic effects.

Occurrence of multidrug resistance associated with extended-spectrum ß­lactamase and the biofilm forming ability of Escherichia coli in environmental swine husbandry.

Extended-spectrum beta-lactamase (ESBL) production and biofilm formation are mechanisms employed by Escherichia coli to resist beta-lactam antibiotics. Thus, we aimed to examine antibiotic resistance associated with ESBL production and biofilm formation in E. coli isolates from swine farms in Southern Thailand. In total, 159 E. coli isolates were obtained, with 44 isolates identified as ESBL producers, originating from feces (18.87 %) and wastewater (8.80 %) samples. All ESBL-producing strains exhibited resistance to ampicillin (100 %), followed by the cephalosporin group (97.73 %) and tetracycline (84.09 %). Multidrug resistance was observed in 17 isolates (38.63 %). Among the isolates from feces samples, the blaGES gene was the most prevalent, detected in 90 % of the samples, followed by blaCTX-M9 (86.67 %) and blaCTX-M1 (66.67 %), respectively. In the bacteria isolated from wastewater, both blaGES and blaCTX-M9 genes were the predominant resistance genes, detected in 100 % of the isolates, followed by blaCTX-M1 (64.29 %) and blaTEM (50 %), respectively. Strong biofilm formation was observed in 11 isolates (36.67 %) from feces and 4 isolates (25.57 %) from wastewater samples. Notably, nearly 100 % of ESBL-producing strains isolated from feces tested positive for both pgaA and pgaC genes, which play a role in intracellular adhesion and biofilm production. These findings contribute to the understanding and potential control of ESBL-producing E. coli, and the dissemination of antibiotic resistance and biofilm-related genes in swine farms.

Acinetobacter baumannii in suspected bacterial infections: Association between multidrug resistance, virulence genes, & biofilm production.

BACKGROUND OBJECTIVES: Acinetobacter baumannii has emerged as a nosocomial pathogen with a tendency of high antibiotic resistance and biofilm production. This study aimed to determine the occurrence of A. baumannii from different clinical specimens of suspected bacterial infections and furthermore to see the association of biofilm production with multidrug resistance and expression of virulence factor genes in A. baumannii. METHODS: A. baumannii was confirmed in clinical specimens by the detection of the blaOXA-51-like gene. Biofilm production was tested by microtitre plate assay and virulence genes were detected by real-time PCR. RESULTS: A. baumannii was isolated from a total of 307 clinical specimens. The isolate which showed the highest number of A. baumannii was an endotracheal tube specimen (44.95%), then sputum (19.54%), followed by pus (17.26%), urine (7.49%) and blood (5.86%), and <2 per cent from body fluids, catheter-tips and urogenital specimens. A resistance rate of 70-81.43 per cent against all antibiotics tested, except colistin and tigecycline, was noted, and 242 (78.82%) isolates were multidrug-resistant (MDR). Biofilm was detected in 205 (66.78%) with a distribution of 54.1 per cent weak, 10.42 per cent medium and 2.28 per cent strong biofilms. 71.07 per cent of MDR isolates produce biofilm (P<0.05). Amongst virulence factor genes, 281 (91.53%) outer membrane protein A (OmpA) and 98 (31.92%) biofilm-associated protein (Bap) were detected. Amongst 100 carbapenem-resistant A. baumannii, the blaOXA-23-like gene was predominant (96%), the blaOXA-58-like gene (6%) and none harboured the blaOXA-24-like gene. The metallo-ß-lactamase genes blaIMP-1 (4%) and blaVIM-1(8%) were detected, and 76 per cent showed the insertion sequence ISAba1. INTERPRETATION CONCLUSIONS: The majority of isolates studied were from lower respiratory tract specimens. The high MDR rate and its positive association with biofilm formation indicate the nosocomial distribution of A. baumannii. The biofilm formation and the presence of Bap were not interrelated, indicating that biofilm formation was not regulated by a single factor. The MDR rate and the presence of OmpA and Bap showed a positive association (P<0.05). The isolates co-harbouring different carbapenem resistance genes were the predominant biofilm producers, which will seriously limit the therapeutic options suggesting the need for strict antimicrobial stewardship and molecular surveillance in hospitals.

Piper betle extract and its application in bovine teat dipping solution inhibit and eliminate biofilms in bovine mastitis-inducing staphylococci.

Background and Aim: Staphylococci, including Staphylococcus aureus, Staphylococcus chromogenes, and Staphylococcus haemolyticus, are significant bacteria that induce bovine mastitis, primarily because they can form biofilms in bovine teat canals. This study aimed to investigate the efficacy of Piper betle extract and a bovine teat dipping solution containing P. betle extract (BSP) against these mastitis-causing staphylococci. Materials and Methods: BSP was prepared using P. betle extract as the bioactive compound. The antibacterial activity of the plant extract and BSP against the pathogens was investigated using a broth microdilution method. The activity of the extract and BSP against the pathogen biofilms was also determined. A stability test was performed to observe the pH, color, turbidity, homogeneity, precipitation, and separation of BSP stored at 4°C and 25°C for up to 4 weeks. Results: The extract exhibited potent antibacterial activity against S. aureus and S. haemolyticus, with similar values for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) ranging from 0.03 mg/mL to 0.125 mg/mL. The MIC and MBC values of the extract against S. chromogenes were 0.5-1 mg/mL and 0.5-2 mg/mL, respectively. Moreover, BSP exhibited MIC and MBC values of 12.5-50 v/v against all tested staphylococci isolates. When used at 1/2 and 1/4 × MIC, the extract and BSP significantly inhibited the formation of staphylococcal biofilms (p < 0.05) in the tested strains. The results indicated that treatment with 1/2 × MIC of the extract and BSP resulted in biofilm inhibition ranging from 30%-66% and 19%-39%, respectively. Furthermore, the extract at 16 × MIC eliminated 54%-86% of established mature isolate biofilms, whereas BSP removed 41%-61% of mature biofilm viability. Storage of BSP at 4°C did not change the factors associated with stability from the 1st to 4th week. Conclusion: These findings suggest that BSP may exhibit potential medicinal benefits in inhibiting the growth and biofilm formation of mastitis-inducing staphylococci in bovines.

Antibacterial and antivirulence factor activities of protein hydrolysates from Phatthalung Sangyod rice (Oryza sativa L.) seeds against zoonotic and foodborne pathogens.

Background and Aim: Antimicrobial resistance is an emerging public health threat. Foodborne illnesses are typically caused by bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus, which are frequently resistant to common antimicrobial agents. Rice is a staple grain in most parts of the world. Our previous work showed that Phatthalung Sangyod rice seed protein hydrolysates (SYPs), especially SYP4, exhibit antifungal activity against several fungal species that are pathogenic for both humans and animals and are non-cytotoxic to animal red blood cells. In this study, we aimed to determine the effects of the bioactive peptides in SYPs against several pathogenic bacteria in humans and animals. Materials and Methods: After isolating SYP1, it was treated as follows: heated (SYP2), and hydrolyzed using pepsin (SYP3), and proteinase K (SYP4). Then, we used 500 µg of protein to evaluate the antibacterial effects on four pathogenic bacteria, including E. coli, P. aeruginosa, B. cereus, and S. aureus, using agar well diffusion. Using a broth microdilution assay, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) values of active SYPs. Using the agar well diffusion and microtube incubation methods, we also assessed the inhibitory effects of SYPs on the bacterial quorum sensing (QS) activity of Chromobacterium violaceum. Sangyod rice seed protein hydrolysates were evaluated for their ability to inhibit the biofilm formation of bacterial cells by a crytal violet assay. Furthermore, using the dropping method, we tested the inhibitory effects of SYPs on the bacterial pigments pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Results: Our results showed that the crude protein lysate (SYP1) did not exhibit antibacterial activity against any of the test bacteria. Intriguingly, after boiling (SYP2) and enzymatic hydrolysis (SYP3 and SYP4), the protein hydrolysates were transformed into bioactive peptides and displayed antibacterial properties against all of the test bacteria at a concentration of 500 µg as determined by agar well diffusion. SYP4 demonstrated the highest antibacterial activity as it completely inhibited all test strains, with inhibition zones ranging from 16.88 ± 0.25 to 21.25 ± 0.5 mm, and also yielded the highest MIC/MBC values against P. aeruginosa, B. cereus, and E. coli, at 256 and >256 µg/mL, respectively. We observed that at least 256 µg/mL of SYP4 is required to exhibit optimal antibacterial activity. At 16-128 µg/mL, it exhibited antibiofilm activity against S. aureus. Furthermore, at 256 µg/mL, SYP4 inhibited pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Although SYP2 and SYP3 displayed weak antibacterial activity and their MIC values could not be obtained for all bacteria, they showed strong QS inhibition in C. violaceum at 256 µg protein. Moreover, SYP2 and SYP3, at a minimum concentration of 32 µg/mL, significantly reduced violacein production. SYP3 also showed biofilm reduction activity on S. aureus at least 16-512 µg/mL. Conclusion: Sangyod Phatthalung protein hydrolysates exerted excellent inhibitory effects against the growth of bacteria and their virulence factors, such as QS, biofilm formation, and/or pigment production. These factors include zoonotic and foodborne pathogens. Therefore, daily consumption of Sangyod Phatthalung rice might reduce the risk of bacterial pathogenesis and foodborne diseases. In conclusion, functional foods or alternate methods of treating bacterial illnesses may be developed in humans and animals.

Antifungal activity of protein hydrolysates from Thai Phatthalung Sangyod rice (Oryza sativa L.) seeds.

Background and Aim: Fungal zoonoses are an economic and public health concern because they can cause various degrees of morbidity and mortality in animals and humans. To combat this issue, alternative natural antifungals, such as products derived from rice protein hydrolysates or rice antifungal protein/peptide are being considered because they are highly bioactive and exhibit various functional properties. Thailand is a leading rice producer and exporter. Among the various cultivated rice varieties, Sangyod rice (Oryza sativa L.) is exclusively indigenous to Thailand's Phatthalung province; it has a Thai geographical indication tag. Here, we investigated whether the Phatthalung Sangyod rice seeds have bioactive antifungal peptides. Materials and Methods: Antifungal activity in four Sangyod rice seed extracts (SYPs) - namely, (1) the crude lysate, SYP1; (2) the heat-treated lysate, SYP2; (3) the heat- and pepsin digested lysate, SYP3; and (4) the heat- and proteinase K-digested lysate, SYP4 - was analyzed. Protein concentrations in these SYPs were determined using the Bradford assay. The total phenolic compound content was determined using the modified Folin-Ciocalteu method in a 96-well microplate. Then, the SYP protein pattern was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, using the agar well diffusion method, the antifungal properties of these SYPs were tested against ten medically important pathogenic fungi. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration values were determined for the active SYPs - SYP2-4. Finally, the clinical safety of SYP4 was determined using a hemolytic assay (using canine red blood cells [RBCs]). Results: The crude lysate SYP1 did not show antifungal activity against any of the ten tested pathogenic fungi. Surprisingly, hydrolysates SYP2, SYP3, and SYP4 displayed antifungal properties against the ten tested pathogenic fungi. Thus, heat and enzymatic hydrolysis seem to transform the bioactivity of the crude protein extract - SYP1. Further, SYP4 shows the most effective antifungal activity. It completely inhibited Cryptococcus neoformans, Talaromyces marneffei yeast phase, Trichophyton mentagrophytes, and Trichophyton rubrum. A partial inhibitory action on Candida albicans and Microsporum gypseum was possessed while showing the least activity to C. neoformans. SYP4 was nontoxic to canine RBCs. Hemolysis of canine RBCs was undetectable at 1 × MIC and 2 × MIC concentrations; therefore, it can be safely used in further applications. Conclusion: These results indicate that heat and proteinase K hydrolyzed SYP is a very potent antifungal preparation against animal and human fungal pathogens and it can be used in future pharmaceuticals and functional foods.

Development of a microencapsulated probiotic containing Pediococcus acidilactici WU222001 against avian pathogenic Escherichia coli.

Background and Aim: Probiotics are beneficial microorganisms for humans and animals. In this study, we developed a microencapsulated probiotic with antibacterial activity against avian pathogenic Escherichia coli (APEC). Materials and Methods: Alignment of the 16S rRNA sequences of the isolate WU222001 with those deposited in GenBank revealed that the isolate was Pediococcus acidilactici with 99.6% homology. This bacterium was characterized as a probiotic based on its tolerance toward in vitro gastrointestinal tract (GIT) conditions, hydrophobicity, and auto-aggregation. The antibacterial activity of the probiotic's culture supernatant against APEC was investigated using a broth microdilution assay. Pediococcus acidilactici was microencapsulated using sodium alginate and agar with diameters ranging from 47 to 61 µm. Then, physicochemical characteristics and stability of the microcapsules were determined. Results: The isolate was characterized as a probiotic based on its resistance to low pH, bile salts, and pancreatin, with relative values of 79.2%, 70.95%, and 90.64%, respectively. Furthermore, the bacterium exhibited 79.56% auto-aggregation and 55.25% hydrophobicity at 24 h. The probiotic's culture supernatant exhibited strong antibacterial activity against clinical APEC isolates with minimum inhibitory concentration and minimum bactericidal concentration of 12.5% and 25% v/v, respectively. Microencapsulation-enhanced bacterial viability in GIT compared to free cells. Moreover, 89.65% of the encapsulated cells were released into the simulated intestinal fluid within 4 h. The viable count in microcapsules was 63.19% after 3 months of storage at 4°C. Conclusion: The results indicated that the culture supernatant of P. acidilactici inhibited the growth of APEC. In addition, microencapsulation extends the viability of P. acidilactici under harsh conditions, indicating its potential application in the feed production.

Virulence factors and quorum sensing as targets of new therapeutic options by plant-derived compounds against bacterial infections caused by human and animal pathogens.

The emergence of antibiotic-resistant bacteria and hospital-acquired bacterial infection has become rampant due to antibiotic overuse. Virulence factors are secondary to bacterial growth and are important in their pathogenesis, and therefore, new antimicrobial therapies to inhibit bacterial virulence factors are becoming important strategies against antibiotic resistance. Here, we focus on anti-virulence factors that act through anti-quorum sensing and the subsequent clearance of bacteria by antimicrobial compounds, especially active herbal extracts. These quorum sensing systems are based on toxins, biofilms, and efflux pumps, and bioactive compounds isolated from medicinal plants can treat bacterial virulence pathologies. Ideally, bacterial virulence factors are secondary growth factors of bacteria. Hence, inhibition of bacterial virulence factors could reduce bacterial pathogenesis. Furthermore, anti-virulence factors from herbal compounds can be developed as novel treatments for bacterial infection. Therefore, this narrative review aims to discuss bacterial virulence factors acting through quorum sensing systems that are preserved as targets for treating bacterial infection by plant-derived compounds.

Trypanosoma lewisi in blood of Rattus rattus complex residing in human settlements, Nakhon Si Thammarat, Thailand: Microscopic and molecular investigations.

Trypanosomes are blood parasites infected in various mammals, including rats. The presence of rats in human settlements can increase the chance of Trypanosoma transmission to humans. The molecular study of multispacer in Trypanosoma spp. in naturally infected rodents in Thailand is scanty. The objective of this study was to detect Trypanosoma in the blood of the captured rats in Nakhon Si Thammarat, Thailand, using microscopic and molecular techniques. This was a cross-sectional study conducted in human settlement areas. Ninety-nine blood samples were collected using cardiac puncture. A blood sample was smeared on a glass slide and examined using a compound light microscope and a scanning electron microscope. Moreover, polymerase chain reaction was applied to detect Trypanosoma evansi and T. lewisi in the blood. An additional primer set was used to confirm the species of the detected trypanosome. Approximately 18% of the rats had positive Trypanosoma infections. All Trypanosoma-positive blood samples were matched with sequences of T. lewisi. The stumpy form of trypanosome had higher nucleus related parameters than the slender form. Interestingly, the partial sequences of the alpha-tubulin gene of T. lewisi were first reported in the naturally infected RrC in this study. Based on the results obtained, T. lewisi biology, particularly the virulent components and route of transmission, pathogenesis, and in vitro experiments, are strongly recommended for further study.

Knema retusa is antibacterial and antibiofilm against antibiotic resistant Staphylococcus aureus and S. haemolyticus isolated in bovine mastitis.

This study aimed to assess antibacterial activity of Knema retusa wood extract (KRe) against antibiotic resistant staphylococci which are causative agents of bovine mastitis. From 75 cases of intramammary infections in dairy cows, 66 staphylococcal isolates were collected, including 11 Staphylococcus aureus isolates (17%) and 55 coagulase-negative staphylococci (83%). Sixty isolates (91%) formed strong biofilms. KRe had minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) against the isolates ranging 32-256 ug/mL and 64-512 ug/mL, respectively. Two-hour KRe exposures at 4×MIC, viabilities of S. aureus and S. haemolyticus decreased by 3 log10 compared to the control. Scanning EM (SEM) showed that KRe disrupted the bacterial cells of both species. KRe at 1/16×MIC significantly inhibited biofilm formation (P < 0.05) in both S. aureus and S. haemolyticus. At 1/2×MIC, S. aureus and S. haemolyticus biofilm inhibition ranged from 75 to 99%. Cells within established biofilms were disrupted 66-83% by KRe at 32×MIC. Moreover, 1/2×MIC KRe reduced bacterial adhesion to glass surfaces observed by SEM. According to GC-MS analysis, the major compound in KRe was endo-2-hydroxy-9,9-(ethylenedioxy)-1-carbethoxy bicyclo [3.3.1] nonane (E2N). Molecular docking analysis of E2N has a high affinity for staphylococcal accessory regulator A (SarA), binding free-energy - 6.40kcal/mol. The results suggested that KRe may have medicinal benefits by inhibiting the growth, biofilm, and adhesion of antibiotic resistant staphylococci isolated from bovine mastitis.

Green synthesis of nano-liposomes containing Bunium persicum and Trachyspermum ammi essential oils against Trichomonasvaginalis.

BACKGROUND: Trichomonas vaginalis, a parasitic flagellated protozoan, is one of the main non-viral sexually transmitted diseases worldwide. Treatment options for trichomoniasis are limited to nitroimidazole compounds. However, resistance to these drugs has been reported, which requires the development of new anti-Trichomonas agents that confer suitable efficacy and less toxicity. METHODS: In the present work, we assessed the effectiveness of the liposomal system containing essential oils of Bunium persicum and Trachyspermum ammi against T. vaginalis in vitro. The chemical composition of B. persicum and T. ammi were analyzed using gas chromatography-mass spectrometry (GC-MS). Liposomal vesicles were prepared with phosphatidylcholine) 70%) and cholesterol)30%) using the thin-film method. The essential oils of B. persicum and T. ammi were loaded into the liposomes using the inactive loading method. Liposomal vesicles were made for two plants separately. Their physicochemical features were tested using Zeta-Sizer, AFM and SEM. The anti-Trichomonas activity was determined after 12 and 24 h of parasite cultures in TYI-S-33 medium. RESULTS: After 12 and 24 h of administration, the IC50 of the B. persicum essential oil nano-liposomes induced 14.41 µg/mL and 45.19 µg/mL, respectively. The IC50 of T. ammi essential oil nano-liposomes induced 8.08 µg/mL and 25.81 µg/mL, respectively. CONCLUSIONS: These data suggested that nano-liposomes of the essential oils of B. persicum and T. ammi may be a promising alternative to current treatments for Trichomonas infection.

Anti-Acanthamoeba activity of a semi-synthetic mangostin derivative and its ability in removal of Acanthamoeba triangularis WU19001 on contact lens.

Garcinia mangostana L., also known as the mangosteen tree, is a native medicinal plant in Southeast Asia having a wide variety of pharmacologically active compounds, including xanthonoid mangostin. In this study, we examined the pharmacological activities of the selected semi-synthetic mangostin derivative, namely, amoebicidal activity, encystation inhibition, excystation activity, and removal capacity of adhesive Acanthamoeba from the surface of contact lens (CL). Among the three derivatives, C1 exhibited promising anti-Acanthamoeba activity against Acanthamoeba triangularis WU19001 trophozoites and cysts. SEM images displayed morphological changes in Acanthamoeba trophozoites, including the loss of acanthopodia, pore formation in the cell membrane, and membrane damage. In addition, the treated cyst was shrunken and adopted an irregular flat cyst shape. Under a fluorescence microscope, acridine orange and propidium iodide (AO/PI) staining revealed C1 induced condensation of cytoplasm and chromatin with the loss of cell volume in the treated trophozoites, while calcofluor white staining demonstrated the leakage of cell wall in treated cysts, leading to cell death. Interestingly, at the concentration ranges in which C1 showed the anti-Acanthamoeba effects (IC50 values ranging from 0.035-0.056 mg/mL), they were not toxic to Vero cells. C1 displayed the highest inhibitory effect on A. triangularis encystation at 1/16×MIC value (0.004 mg/mL). While C1 demonstrated the excystation activity at 1/128×MIC value with a high rate of 89.47%. Furthermore, C1 exhibited the removal capacity of adhesive Acanthamoeba from the surface of CL comparable with commercial multipurpose solutions (MPSs). Based on the results obtained, C1 may be a promising lead agent to develop a therapeutic for the treatment of Acanthamoeba infections and disinfectant solutions for CL.

Robusta coffee extracts inhibit quorum sensing activity in Chromobacterium violaceum and reduce biofilms against Bacillus cereus and Staphylococcus aureus.

Background and Aim: Bacillus cereus and Staphylococcus aureus cause foodborne intoxication in humans and animals. Pathogens can produce biofilms controlled by the quorum sensing system. The study aimed to investigate the antibacterial, antibiofilm, and anti-quorum sensing activities of Coffea canephora P. ex Fr. (Robusta coffee) extracts against B. cereus and S. aureus. Materials and Methods: Ethanol extracts of fruit peels and seeds of Robusta coffee were tested for antibacterial activity against B. cereus and S. aureus using a broth microdilution assay. Reduction of the biofilm formation and elimination of the viability of mature biofilm-grown cells of B. cereus and S. aureus were determined. Inhibition of quorum sensing activity in Chromobacterium violaceum by the extracts was investigated using the disk diffusion method and flask incubation assay. Results: Fresh fruit peel extract showed the strongest antibacterial activity against B. cereus and S. aureus with minimum inhibitory concentration (MIC) values of 2 and 4 mg/mL, respectively. However, the extracts did not inhibit Escherichia coli, avian pathogenic E. coli, and Pseudomonas aeruginosa at 8 mg/mL. Significant inhibition of biofilm formation at 1/2 × MIC of the fresh peel extract was detected in B. cereus (56.37%) and S. aureus (39.69 %), respectively. At 8 × MIC of the fresh peel extract, a significant elimination of the mature biofilm viability was detected in B. cereus (92.48%) and S. aureus (74.49%), respectively. The results showed that fresh and dried peel fruit extracts at 1/2 × MIC significantly reduced violacein production with the highest percentage inhibition ranging from 44.53 to 47.48% at 24 h (p ≤ 0.05). Conclusion: The results of the present study suggest the potential therapeutic benefits of Robusta coffee extracts in inhibiting the growth, biofilm, and quorum sensing of both B. cereus and S. aureus. The results put forward an alternative strategy to control the foodborne intoxications caused by both pathogens.

Sangyod rice bran extract enhances Lacticaseibacillus paracasei growth during the exponential phase and antibacterial activity of L. paracasei supernatant against zoonotic and foodborne pathogens.

Background and Aim: Prebiotics are a group of nutrients or compounds that are degraded by the gut microbiota, including Lacticaseibacillus paracasei. The probiotic plays an important role in adhesion to the gut and is able to produce antimicrobial substances to inhibit pathogens. This study aimed to investigate the effects of Sangyod rice bran extract on the growth promotion of L. paracasei. Furthermore, antibacterial activity of the extract and L. paracasei supernatants cultured in De Man, Rogosa and Sharpe (MRS) medium plus the extract against zoonotic and foodborne pathogens was investigated. Materials and Methods: Antibacterial activity of the crude extract and the oil from Sangyod rice bran against the pathogens, including Bacillus cereus, Staphylococcus aureus, Escherichia coli, Avian pathogenic E. coli, and Pseudomonas aeruginosa was investigated using broth microdilution assay. The effects of the crude extract and the oil on the growth and adhesion of L. paracasei were further determined. The antibacterial activity of L. paracasei supernatant cultured in the medium supplemented with the extract and the oil against the pathogens was determined by agar well diffusion assay, followed by the broth microdilution assay. Finally, the chemical constituents and antioxidant activity of the crude extract and the oil from Sangyod rice bran were investigated. Results: The crude extract and the oil from Sangyod rice bran enhanced L. paracasei growth during the exponential phase. Furthermore, the crude extract at 0.25 mg/mL significantly enhanced the adhesion of L. paracasei to the surface compared with the control. Both minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of the crude extract against B. cereus and S. aureus were 0.5 and 1.0 mg/mL, respectively. All pathogens were sensitive to the supernatant of L. paracasei with similar MIC and MBC ranging from 12.5% v/v to 50% v/v. However, the MIC and MBC values of L. paracasei supernatant grown in MRS medium plus the crude extract and oil were not significantly different compared to the supernatant obtained from MRS alone. The crude extract had free radical scavenging activities with IC50 values at 0.61 mg/mL. Conclusion: The results suggested the potential benefits of the crude extract from Sangyod rice bran for inducing the growth and the adhesion of L. paracasei and inhibiting zoonotic and foodborne pathogens.