Whole genome sequencing analysis on antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia.

IMPORTANCE: The emergence and rapid increase in the incidence of multidrug-resistant (MDR) bacteria in pig farms has become a serious concern and reduced the choice of effective antibiotics. OBJECTIVE: This study analyzed the phylogenetics and diversity of antibiotic resistance genes (ARGs) and molecularly identified the source of ARGs in antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia. METHODS: Forty-four antibiotic-resistant E. coli isolates from fecal samples from 44 pig farms in Banten Province, Indonesia, were used as samples. The samples were categorized into 14 clusters. Sequencing was performed using the Oxford Nanopore Technologies MinION platform, with barcoding before sequencing with Nanopore Rapid sequencing gDNA-barcoding (SQK-RBK110.96) according to manufacturing procedures. ARG detection was conducted using ResFinder, and the plasmid replicon was determined using PlasmidFinder. RESULTS: Three phylogenetic leaves of E. coli were identified in the pig farming cluster in Banten Province. The E. coli isolates exhibited potential resistance to nine classes of antibiotics. Fifty-one ARGs were identified across all isolates, with each cluster carrying a minimum of 10 ARGs. The ant(3'')-Ia and qnrS1 genes were present in all isolates. ARGs in the E. coli pig farming cluster originated mainly from plasmids, accounting for an average of 89.4%. CONCLUSIONS AND RELEVANCE: The elevated potential for MDR events, coupled with the dominance of ARGs originating from plasmids, increases the risk of ARG spread among bacterial populations in animals, humans, and the environment.

Detection of tetracycline resistance genes and their diversity in Escherichia coli isolated from pig farm waste in Banten province, Indonesia.

Background and Aim: Livestock waste in the form of feces and liquid represents an important reservoir of antibiotic resistance genes (ARGs). Because many ARGs can be horizontally transferred to other pathogens, livestock waste plays an essential role in the emergence and transmission of various ARGs in the environment. Therefore, this study aimed to detect and assess the diversity of tet genes in Escherichia coli isolated from pig farm waste in Banten province, Indonesia. Materials and Methods: Solid waste (feces) and wastewater were collected from 44 pig farms in Banten province. The isolation and identification of E. coli referred to the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli World Health Organization (2021) guidelines. tet genes were detected using quantitative real-time polymerase chain reaction after dividing pig farms in the province into four clusters based on their adjacent areas and characteristics. Results: tetA, tetB, tetC, tetM, tetO, and tetX were detected in solid waste and wastewater from pig farms, whereas tetE was not detected in either sample type. tetX (100%) and tetO (75%) were the most dominant genes in solid waste, whereas wastewater samples were dominated by tetA, tetM, tetO, and tetX (prevalence of 50% each). Furthermore, eight tet gene patterns were found in pig farm waste (prevalence of 12.5% each). Conclusion: The results showed a high prevalence of tetO and tetX in solid waste and wastewater from pig farms in Banten province. This significant prevalence and diversity indicated the transmission of tet genes from pigs to the environment, posing a serious threat to public health.

Purification and proteomic analysis of potent fibrinolytic enzymes extracted from Lumbricus rubellus.

BACKGROUND: Lumbrokinase derived from earthworms, Lumbricus rubellus is known to have fibrinolytic enzymes that have potential as therapeutic drugs due to its ability to dissolve fibrin. The current study is aimed to purify the Lumbrokinase from L. rubellus and identify its protein component. METHODS: Water extract of local earthworm Lumbricus rubellus revealed several proteins. Therefore, to identify its protein component, purification through HiPrep DEAE fast flow and proteomic analysis were conducted prior to identifications. A combination of two-dimension gel electrophoresis (2DE) and electrospray ionization mass spectrometry analysis was used to identify the purified fractions. RESULTS: The purified fractions contain five protein bands, namely F25-1, F25-2, F85-1, F85-2, and F85-3, which displayed strong fibrinogenolytic activity. F25 fractions showed fibrinogenolytic activity of 974.85 U/mg, while F85 fractions showed higher activity of 1,484.11 U/mg. Fractions F85-1, F85-2, and F85-3 showed molecular weights of 42.6 kDa, 27.03 kDa, and 14 kDa, respectively and were identified as Lumbrokinase iso-enzymes. CONCLUSION: This preliminary study indicates that the F25 and F85 fractions are similar to published fibrinolytic protease-1 and lumbrokinase, respectively, in terms of their amino acid sequence.

Distribution analysis of tetracycline resistance genes in Escherichia coli isolated from floor surface and effluent of pig slaughterhouses in Banten Province, Indonesia.

Background and Aim: Slaughterhouses and their effluents could serve as a "hotspot" for the occurrence and distribution of antibiotic-resistant bacteria in the environment. This study aimed to understand the distribution of tetracycline resistance genes in Escherichia coli isolated from the floor surface and effluent samples of pig slaughterhouses in Banten Province, Indonesia. Materials and Methods: Ten samples, each from floor surface swabs and effluents, were collected from 10 pig slaughterhouses in Banten Province. Escherichia coli strains were isolated and identified by referring to the protocol of the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli from the WHO (2021). Quantitative real-time polymerase chain reaction (qPCR) was used to detect the tet genes. Results: The tetA, tetB, tetC, tetM, tetO, and tetX genes were distributed in the isolates from the floor surface samples, and the tetA, tetC, tetE, tetM, tetO, and tetX genes were distributed in the isolates from the effluent samples. The tetO gene (60%) was the most dominant gene in the isolates from floor surface samples, while the tetA gene was the dominant one in the isolates from the effluent samples (50%). The tetA + tetO gene combination was the dominant pattern (15%) in the E. coli isolates. Conclusion: The high prevalence and diversity of the tet genes in floor surface and effluent samples from pig slaughterhouses in Banten Province indicated that the transmission of the tet genes had occurred from pigs to the environment; thus, this situation should be considered a serious threat to public health.

A preliminary metagenomics study of bacteria present in the dirt of Swiftlet farmhouses based on nitrite levels in edible bird's nest on Sumatera Island, Indonesia.

Background and Aim: Since the past decade, metagenomics has been used to evaluate sequenced deoxyribonucleic acid of all microorganisms in several types of research. Nitrite contamination originates from the natural environment in Swiftlet farmhouses (SFHs) and can influence nitrite levels in edible bird's nest (EBN). It is strongly speculated that the conversion process into nitrite is influenced by the bacteria present in SFHs. Nitrite can cause adverse effects on human health. The previous research has focused on the characteristics of bacteria that may influence the nitrite conversion process in SFHs. This study aimed to a metagenomics analysis of bacteria present in the dirt of SFHs and evaluated nitrite levels in EBN on Sumatera Island. Materials and Methods: In total, 18 SFHs on Sumatera Island were selected, and EBN and dirt samples were collected from each SFH, resulting in 18 EBN and 18 dirt SFH samples. Raw uncleaned white EBN and dirt from three areas of SFH were collected. The samples were analyzed for nitrite levels using a spectrophotometer, and the metagenomics sequencing of SFH dirt samples was performed using the MinIon nanopore method. The sequenced data were analyzed using the EPI2ME software. Results: Of the 18 raw uncleaned white EBN samples, 9 (50%) had <30 ppm nitrite levels. The top five bacterial genera in SFH dirt samples in Group A (nitrite levels >30 ppm) were Aeromonas, Escherichia, Acinetobacter, Arcobacter, and Acetoanaerobium. Those in Group B (nitrite levels <30 ppm) were Aeromonas, Pseudomonas, Shewanella, Escherichia, and Acinetobacter. There were 12 genera of nitrifying bacteria in Group A and 8 in Group B. The total cumulative read of nitrifying bacteria in Groups A and B were 87 and 38 reads, respectively. Conclusion: This is the first study to show that characteristic bacteria present in the dirt of SFHs might significantly influence the conversion from nitrogen to nitrite. Approximately 50% of raw uncleaned EBN samples had <30 ppm nitrite levels. Aeromonas was the most dominant bacterial genus found in Groups A and B. The variations in genus and cumulative reads nitrifying bacteria in group A were greater than those in Group B. This study provides information on the characteristics of bacteria that may influence the nitrite conversion process in SFHs. Metagenomics data were obtained from the reading using the software EPI2ME. Further research is needed on the bacterial target species that can convert nitrite in SFHs.

The association between FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a among chronic granulocytic leukemia patients treated with imatinib mesylate.

Background: The gene  FOXO3a has been elucidated to govern the development of chronic granulocytic leukemia (CGL). Moreover, it has been suggested that the levels of  FOXO3a in circulation are affected by the  FOXO3a rs4946936 gene polymorphism. However, no study has assessed the correlation between the  FOXO3a rs4946936 gene polymorphism and the levels of  FOXO3a. The objective of this study was to assess the association between the  FOXO3a rs4946936 gene polymorphism and the levels of  FOXO3a in CGL patients treated with imatinib mesylate.  Methods: A cross-sectional study was conducted from February 2019 to February 2020. The genotyping of  FOXO3a rs4946936 gene polymorphism was conducted using PCR-RFLP, and the levels of  FOXO3a were assessed using ELISA. The association between the  FOXO3a rs4946936 gene polymorphism and the levels of  FOXO3a were assessed using multiple logistic regression.  Results: A total of 60 CGL patients were assessed in our study. Among them, the CC, CT, and TT genotypes of the  FOXO3a rs4946936 gene polymorphism were 35.0%, 48.3%, and 16.7% respectively. Our calculation revealed that elevated levels of  FOXO3a were found in CGL patients with the CC genotype of the  FOXO3a rs4946936 gene polymorphism. While we failed to clarify the association between either the CT or the TT genotype of  FOXO3a rs4946936 gene polymorphism and the levels of  FOXO3a.  Conclusion: Our study identifies that the CC genotype of the  FOXO3a rs4946936 gene polymorphism affects the elevated levels of  FOXO3a in CGL patients treated with imatinib mesylate.

Identification and differentiation of Campylobacter isolated from chicken meat using real-time polymerase chain reaction and high resolution melting analysis of hipO and glyA genes.

BACKGROUND AND AIM: Campylobacter species have been recognized as the most frequently identified bacterial cause of human gastroenteritis. The aims of this study were to identify Campylobacter jejuni and Campylobacter coli species isolated from chicken meat and to analyze the differences in the melting curve patterns of both species. MATERIALS AND METHODS: A total of 105 chicken meat samples collected from slaughterhouses and retailers in six provinces in Indonesia were examined for the isolation and identification of Campylobacter spp. A total of 56 positive isolates of Campylobacter spp. were analyzed using the quantitative real-time polymerase chain reaction and high resolution melting method. RESULTS: The prevalence of Campylobacter spp. in chicken meat was found to be 61.9%. Regarding the identification, 23 isolates (41.07%) were C. jejuni, 22 (39.29%) were C. coli, six (10.71%) were a mix between C. jejuni and C. coli, and five isolates (8.93%) were Campylobacter spp. All the C. jejuni and C. coli isolates produced varied melting curve patterns. CONCLUSION: The high prevalence of C. jejuni and C. coli in chicken meat in Indonesia indicates a high risk of the incidence of campylobacteriosis in humans.

Green isolation and physical modification of pineapple stem waste starch as pharmaceutical excipient.

The waste of inedible parts of pineapple, particularly in tropical countries, contributes to environmental burden. This study aimed to utilize pineapple stem waste as a source of starch-based pharmaceutical excipient. The starch was isolated from pineapple stem waste using a simple process without applying harsh chemicals. The isolated starch (PSS) was then physically modified through gelatinization and spray drying to improve its physical properties. Starch characteristics were identified by FTIR, TGA, and XRD analysis. The SEM imaging showed morphological change with reduced surface roughness due to physical modification of the starch. Decreased crystallinity of modified starch (MPS) was confirmed by our XRD results: the peaks of A-type crystalline at 2θ of 13°, 15°, 18°, and 23° were present in PSS, yet mostly absent in MPS. Thermogravimetric analysis showed that MPS behaved differently from PSS and the degradation events occurred at lower temperature. When the starch was spray-dried without prior gelatinization process, the physicochemical characteristics of spray-dried starch resembled untreated starch. Moisture content in PSS (10.66%) decreased after gelatinization to 7.3%. Potential use of MPS was demonstrated by its powder flowability (Student's t test, p < 0.05), swelling capacity (Student's t test, p < 0.05), and compaction profile. In summary, our findings demonstrated that modified pineapple starch showed better physical characteristics and quite promising as a tablet binder and disintegrant.

Tacorin, an extract from Ananas comosus stem, stimulates wound healing by modulating the expression of tumor necrosis factor α, transforming growth factor ß and matrix metalloproteinase 2.

Wound healing is a complex biological process that involves integration of hemostasis, inflammation, proliferation and tissue remodeling. An extract of pineapple (Ananas comosus) stem demonstrates several therapeutics properties, including acceleration of wound healing. Tacorin is a water crude extract derived from the stem of A. comosus with high protein content. The effect of tacorin on wound healing in vivo was examined using rats with an induced injury. Wound closure was faster with tacorin treatment than in the untreated group. An in vitro study was conducted on mammalian cells (3T3-L1) to observe the effect of tacorin on cell proliferation. Tacorin was first heated to inactivate its proteolytic activity. It increased the viability of 3T3-L1 cells in a dose-dependent manner. Excessive inflammation was suppressed by tacorin as shown by decreased tumor necrosis factor α expression. Treatment with tacorin increased the expression of transforming growth factor ß, a major player in tissue remodeling. Moreover, tacorin also reduced the expression of MMP-2 to accelerate the recovery of the wound. Taken together, tacorin is able to accelerate the wound-healing process by increasing cell proliferation, suppressing inflammation and accelerating tissue remodeling.

Bioactive protein fraction DLBS1033 containing lumbrokinase isolated from Lumbricus rubellus: ex vivo, in vivo, and pharmaceutic studies.

DLBS1033 is a bioactive protein fraction isolated from Lumbricus rubellus that tends to be unstable when exposed to the gastrointestinal environment. Accordingly, appropriate pharmaceutical development is needed to maximize absorption of the protein fraction in the gastrointestinal tract. In vitro, ex vivo, and in vivo stability assays were performed to study the stability of the bioactive protein fraction in gastric conditions. The bioactive protein fraction DLBS1033 was found to be unstable at low pH and in gastric fluid. The "enteric coating" formulation showed no leakage in gastric fluid-like medium and possessed a good release profile in simulated intestinal medium. DLBS1033 was absorbed through the small intestine in an intact protein form, confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis. This result confirmed that an enteric coating formula using methacrylic acid copolymer could protect DLBS1033 from the acidic condition of the stomach by preventing the release of DLBS1033 in the stomach, while promoting its release when reaching the intestine. From the blood concentration-versus-time curve, (99m)Tc-DLBS1033 showed a circulation half-life of 70 minutes. This relatively long biological half-life supports its function as a thrombolytic protein. Thus, an enteric delivery system is considered the best approach for DLBS1033 as an oral thrombolytic agent.

Antibodies against dengue virus nonstructural protein-1 induce heme oxygenase-1 via a redox-dependent pathway in human endothelial cells.

Heme oxygenase (HO)-1, the inducible isoform of the first and rate-limiting enzyme of heme degradation, affords anti-inflammatory protection via its cell-type-specific effects in endothelial cells (ECs). In dengue hemorrhagic fever (DHF), which is the life-threatening form of dengue virus (DV) infection, endothelial interactions of cross-reactive antibodies against the DV nonstructural glycoprotein-1 (NS1) are associated with endothelial dysfunction. In this study, we investigated whether anti-NS1 antibodies might regulate HO-1 gene expression in human ECs. Serum from DHF patients with high anti-NS1 titers and a monoclonal anti-NS1 antibody upregulated HO-1 gene expression in human umbilical vein ECs, which was blocked by purified NS1 antigen. Immunoprecipitation studies showed that anti-NS1 antibodies specifically bound to the oxidoreductase protein disulfide isomerase (PDI) on ECs. Moreover, anti-NS1-mediated HO-1 induction was reduced by inhibition of PDI enzyme activity. Reactive oxygen species, which were generated by NADPH oxidase and in turn activated the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, were involved in this upregulation of HO-1 gene expression. Finally, apoptosis of ECs caused by anti-NS1 antibodies was increased by pharmacological inhibition of HO-1 enzyme activity. In conclusion, HO-1 gene expression is upregulated by anti-NS1 antibodies via activation of a redox-dependent PDI/PI3K/Akt-mediated pathway in human ECs.