Harnessing black soldier fly (Hermetia illucens) prepupae against Aeromonas hydrophila: Fermentation-based fatty acids production and its bioinformatic assessment.

Background: Aeromonas hydrophila (A. hydrophila) is a bacterium with zoonotic potential and is multidrug-resistant. It utilizes hemolysin and aerolysin to spread infection. Black soldier flies (BSFs) can be antibacterial because of the fatty acids it contains. Aims: This study aimed to investigate and compare the fatty acid profiles of BSF prepupae grown in fermented and nonfermented media using bioinformatics tools and assess their potential as antibacterial agents against A. hydrophila. Methods: The study used BSF prepupae reared on various organic substrates. BSF prepupae grown in fermented or nonfermented substrate were observed against fatty acid. The fatty acid analysis was performed using GC-MS. Fatty acids were analyzed statistically using the one-way ANOVA test with a 95% confidence level. Fatty acid bioactivity was predicted using the online PASS-two-way drug program. Molecular docking on BSF fatty acid compounds was analyzed with PyMol 2.2 and discovery Studio version 21.1.1. Results: The molecular docking test showed the strongest bond was oleic acid with aerolysin and linoleic acid with hemolysin. BSF prepupae grown on fermented media showed higher crude fat and saturated fatty acids (SFAs) but lower unsaturated fatty acids than nonfermented media. Conclusion: Black soldier fly prepupae, particularly those grown on fermented media, possess antibacterial activity against A. hydrophila through potential fatty acid-mediated inhibition of crucial virulence factors.

Aeromonas hydrophila induction method in adult zebrafish (Danio rerio) as animal infection models.

Background and Aim: Zebrafish are frequently used as model organisms in scientific research as their genes mirror those of humans. Aeromonas hydrophila bacteria can infect humans and animals, mainly fish. This study aimed to identify the concentration and route of A. hydrophila infection in adult zebrafish. Zebrafish had been used as a challenge test by analyzing their hematological profiles, blood glucose levels, and survival rates. Materials and Methods: Induction of cell supernatant free (CSF) from A. hydrophila bacteria in adult zebrafish was carried out via bath immersion (BI), intraperitoneal injection (IPI), intramuscular injection (IMI), and healthy zebrafish as a control (C). The bacterial concentrations were 107, 109, and 1011 colony-forming units (CFU)/mL. At 24 h post-infection, the outcomes of infection were evaluated based on survival rates, hematological profiles, and blood glucose levels. A one-way analysis of variance with a confidence level of 95% was employed to examine the data. Results: In the BI, IPI, and IMI treatment groups, the survival rate of the fish reached a peak of 100%, 22%-100%, and 16%-63%, respectively, compared with the injection technique. In the IMI2 group, a 109 CFU/mL bacterial concentration was determined to correspond to the lethal dosage 50. All infection groups had lower erythrocyte and hemoglobin counts but higher leukocyte counts than the control group. The blood sugar levels of the healthy and infected groups were not significantly different. Conclusion: The route of A. hydrophila infection through Intramuscular injection with a concentration of 109 CFU/mL indicated a high performance compared to other techniques. This method could be developed as a reproducible challenge test.

Methanol extract of Black soldier fly (Hermetia illucens) prepupae against Aeromonas and Staphylococcus aureus bacteria in vitro and in silico.

Background: Staphylococcus and Aeromonas bacteria are pathogens in humans and animals. The therapy disrupts the virulence structure of the bacteria, resulting in bacterial death. Currently, chemical drugs have resulted in many resistant bacteria, so it is necessary to find alternative natural materials that are not toxic and do not quickly induce resistance. Aims: This study aimed to analyze the potential of methanol extract from Black soldier fly (BSF) prepupae as an antibacterial agent against Staphylococcus aureus and Aeromonas through in silico and in vitro tests. Methods: The BSF prepupae methanol extract was analyzed for protein and fatty acid contents. Disc diffusion method, minimal inhibitory concentration, and minimum bactericidal concentration test were used for in vitro tests against Staphylococcis and Aeromonas. Molecular docking of the active ingredients (defensin, chitin, and chitosan as well as fatty acids) in BSF was downloaded from the NCBI database and docked by the Hex Cuda version 8.0 program with Correlation type parameters Shape + Electro and Grid Dimension version 0.6. Docking results were analyzed using the Discovery Studio program version 21.1.1. Results: The highest fatty acid contents in the extract were palmitic acid and myristic acid. Methanol extract from BSF prepupae acted as a bactericidal agent against S. aureus at a concentration of 320 mg/ml, in contrast to Aeromonas, which still showed bacterial growth. The results of the in silico test showed that defensin-aerolysin and defensin-hemolysin was bound to the same active site area. However, the amount of binding energy produced by 69-Defensin-83-aerolysin was higher than all defensin types in BSF against Aeromonas. Chitin and chitosan showed a bond on the active site of aerolysin and hemolysin, but chitosan had a stronger bond than chitin. In silico study also showed the strongest binding affinity of BSF fatty acids to isoleucyl-tRNA synthetase of S. aureus. Conclusion: The study showed that methanol extract from BSF prepupae had potential capability as an antibacterial agent against S. aureus than Aeromonas in vitro and in silico.

Green Coffea robusta (Coffea canephora) from Lampung province effect toward free radicals in chickens infected with Salmonella enteritidis bacteria.

Background: Foodborne diseases are caused by acquired pathogenic bacteria such as Salmonella enteritidis. It causes an intestinal imbalance and the microbial toxins found in the gastrointestinal tract induce symptoms such as diarrhea. Coffee contains active ingredients such as antioxidants and is used as an anti-inflammatory agent by reducing pro-inflammatory cytokine levels in the body. Aim: The purpose of this study was to determine the interaction between Lampung's robusta coffee and tissue damage in chickens infected by S. enteritidis. Methods: This study used first-day-old Isa brown layer chickens (n = 60), which were divided into five treatment groups. The negative control group consisted of healthy and normal chickens, whereas the positive control group consisted of chickens infected with S. enteritidis bacteria at a concentration of 108 CFU/ml. Groups T1, T2, and T3 were given coffee extract with doses of 500 mg/kg BW (low dose), 1,000 mg/kg BW (moderate dose), and 1,500 mg/kg BW (high dose), respectively, and then infected with S. enteritidis bacteria at a concentration of 108 CFU/ml. The coffee extract and bacteria were given orally via a feeding tube at a volume of 0.5 ml per chick. The extract was given for 14 days (from day 3 to day 16), and the bacteria were given on days 16 and 17. On day 18, the chickens were necropsied. The malondialdehyde (MDA) level was analyzed using one-way analysis of variance test with the GLM procedure (<0.05), while the tissue histopath was analyzed using a descriptive qualitative study to examine the ileal damage. Results: The results showed that the MDA levels (nmol/l) decreased in treatment groups T1, T2, and T3 compared to the positive control. On the contrary, we found improvements in the ileum histopathology of group T1 and T2 in the form of normal and regular intestinal epithelium arrangement of the ileum, long intestinal villi, and decreased total leukocytes. Conclusion: Green coffee robusta has the potential to increase antioxidants and reduce inflammation in the small intestine of chickens infected with S. enteritidis.

Diabetes sepsis on Wistar rat strain (Rattus norvegicus) induced by streptozotocin and bacteria Staphylococcus aureus.

BACKGROUND AND AIM: Sepsis is characterized by loss of control of the inflammatory response, which can be triggered by various microorganisms and toxic secretions. The mortality rate increases due to impaired endothelial function caused dysfunctional organ systems. Diabetes is closely related to sepsis. The study aimed to determine the method of using animal models of sepsis diabetes through a combination of streptozotocin (STZ) and Staphylococcus aureus infection based on biological marker parameters. MATERIALS AND METHODS: A total of 30 male Wistar rats of 2.5-3 months old weighing approximately 150-250 g body weight (BW) divided into six treatment groups with five replications per group were used in the study. Treatment A was negative control (healthy rats) and Treatment B was the positive control (with diabetes) where rats were given STZ dose at 45 mg/kg BW on day 8 intraperitoneally (IP). The blood glucose was measured on day 10, Treatment C was a positive control (bacteria), rats inoculated with S. aureus with a concentration of 108 CFU/mL on day 8 given IP and observed sepsis conditions on day 10th. Treatment group (D, E, and F): Rats given STZ dose at 45 mg/kg BW on day 8th by IP and measured blood glucose on day 10th, then inoculated with S. aureus with different concentrations of 105 CFU/mL, 106 CFU/mL, and 107 CFU/mL on the 10th day, respectively, and were later observed the condition of sepsis on day 12th. Data on diabetes bacteremia were quantitative used blood glucose levels, the bacterial count, and C-reactive protein (CRP) and were analyzed using the one-way analysis of variance test with a confidence level of 95%. Physical examination (temperature and respiration) is qualitative. RESULTS: Physical examination showed that all treatments had a normal temperature, an increased pulse in Groups D, E, and F and a decrease in respiratory rate in the treatment of E and F, the bacteria found in the vital organs in all groups, and CRP levels were not significantly different at all. CONCLUSION: Animal model of diabetes sepsis can be observed through a combination of pancreas damage, and respiration, the bacteria in the vital organs.

Effect of Newcastle disease virus level of infection on embryonic length, embryonic death, and protein profile changes.

BACKGROUND AND AIM: Newcastle disease virus (NDV) is an obligate intracellular parasite. Virus can only live on living cells. The embryonated chicken eggs (ECEs) are one of the growth media of virus that is a cheap, easy to do, and accurate for showing patterns of virus change in the host. Higher virus titers indicate the higher number of viruses and more virulent to infect host. This research aimed to investigate the effect of different level of NDV titer infection in ECEs on protein profile, embryonic length, mortality, and pathological change. MATERIALS AND METHODS: The study used a completely randomized design of six treatments and seven replications. The treatments were different level of NDV titer infection in allantoic fluid (AF) of 9-11 days ECEs, i.e., P1=20, P2=26, P3=27, P4=28, P5=29, and P6=210 hemagglutination unit (HAU). All samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Data were analyzed using one-way ANOVA with p=0.05 for length of the embryo and descriptive analysis for embryo mortality, pathology change, and protein band. RESULTS: The result showed that protein profile of NDV-infected ECEs of all different levels is more complex than protein profile of no NDV-infected ECEs. NDV infected of all different levels showed longer size embryo, higher mortality embryo at the first 2 days, and higher occurrence of hemorrhagic in all part of bodies of embryo than those of no NDV infected. CONCLUSION: It was concluded that NDV infection of all different level decreased health conditions of chicken embryo of ECEs of 9-11 days old. Different level of NDV infection of ECEs of 9-11 days old showed no significantly different embryo profiles. However, all of the NDV-infected embryos were shorter, death on the 2nd day, and suffered more hemorrhage on all body surfaces than uninfected NDV embryos.