Environmental Sample Stability for Pharmaceutical Compound Analysis: Handling and Preservation Recommendations.

Efficient and resilient techniques for handling samples are essential for detecting pharmaceutical compounds in the environment. This study explores a method for preserving water samples during transport before quantitative analysis. The study investigates the stability of 17α-ethynylestradiol (EE2), acetaminophen (ACM), oxytetracycline (OTC), sulfamethoxazole (SMX), and trimethoprim (TMP) after preconcentration within solid-phase extraction (SPE) cartridges. Through various experiments involving different holding times and storage temperatures, it was determined that four pharmaceutical compounds remained stable when stored for a month at 4°C and for six months when stored at -18°C in darkness. Storing these compounds in SPE cartridges at -18°C seemed effective in preserving them for extended periods. In addition, ACM, TMP, OTC, EE2, and SMX remained stable for three days at room temperature. These findings establish guidelines for appropriate storage and handling practices of pharmaceutical compounds preconcentrated from aqueous environmental samples using SPE.

Biotechnologically potential genes in a polysaccharide-degrading epibiont of the Indonesian brown algae Hydroclathrus sp.

BACKGROUND: Marine bacteria have recently attracted increasing attention to be harnessed for the production of valuable enzymes, vitamins, and bioactive compounds. Bacteria associated with the surfaces of marine macroalgae, called epibionts, are particularly interesting from ecological and biotechnological points of view, as they often exhibit antimicrobial activities to compete with pathogenic bacteria for nutrients and spaces. In search for biotechnologically potential genes from marine bacteria, we sequenced and analysed the genome of the epibiont HI03-3b, a polysaccharide-degrading bacterium associated with the surface of the Indonesian brown algae Hydroclathrus sp. RESULTS: The algal epibiont HI03-3b has a genome of approximately 4,860,704 bp in size with 42.02 mol% G + C content, consisting of 5655 open reading frames (ORFs), 4409 genes coding for proteins (CDSs), 94 genes for tRNAs, and 32 genes for rRNAs. The genome sequence of HI03-3b was most closely related to that of Cytobacillus firmus NCTC10335 with the average amino acid identity (AAI) of 95.0 %, average nucleotide identity (ANI) of 94.1 %, and a recommended DNA-DNA hybridization (DDH) of 57.60 %. These scores are lower than the most frequently used standard for species demarcation (95% ANI cutoff) and the new species threshold (DDH > 70.0% for the same bacterial species). Some differences in genome features and gene composition were observed between HI03-3b and NCTC10335, such as genes encoding carbohydrate active enzymes. These suggest that HI03-3b is unique and likely a novel species within Cytobacillus genus, and we therefore proposed its name as Cytobacillus wakatobiense HI03-3b. Genome sequence analyses indicated the presence of genes involved not only in polysaccharide and protein degradation but also in vitamin and secondary metabolite biosynthesis. Some of them encode enzymes and compounds with biotechnological interest, such as protease, chitinase, subtilisin, pullulanase, and bacillolysin, which are often associated with antimicrobial or antibiofilm activities. This antimicrobial potential is supported by our finding that the extracellular protein fraction of this epibiont inhibited the growth of the bacterial pathogen Staphylococcus aureus. CONCLUSION: The epibiont Cytobacillus HI03-3b harbours genes for polysaccharide and protein degradation as well as for natural product biosynthesis, suggesting its potential ecological roles in outcompeting other bacteria during biofilm formation as well as in protecting its algal host from predation. Due to the presence of genes for vitamin biosynthesis, it might also provide the algal host with vitamins for growth and development. Some of these metabolic genes are biotechnologically important, as they could become a platform for bioengineering to generate various seaweed-derived substances sustainably, such as antibiofilm agents and vitamins, which are beneficial for human health.

Emerging contaminants detected in aquaculture sites in Java, Indonesia.

Pharmaceuticals of emerging concern (acetaminophen (ACM), trimethoprim (TMP), oxytetracycline (OTC), and sulfamethoxazole (SMX)) were detected in water samples from aquaculture environments and nonaquaculture sites in four regions located on the northern coast of Central Java. ACM was the most prevalent pharmaceutical, with a mean concentration ranging from not detected (n.d.) to 5.5 ± 1.9 ngL-1 (Brebes). Among the target antibiotics (TMP, OTC, SMX), OTC was the most ubiquitous, with a mean concentration varying from n.d. to 8.0 ± 3.3 ngL-1. Correlation analysis demonstrated that there was a significant correlation between TMP and SMX concentrations. Based on ecological risk assessment evaluation, the use of OTC requires serious consideration, as it presented high health risks to algae, while ACM, TMP, and SMX posed an insignificant to moderate risk to algae, invertebrates, and fish. The findings obtained from this study highlight OTC as an emerging contaminant of prominent concern. More attention needs to be given to managing and planning for the sustainable management of shrimp farms, particularly in the northern part of Central Java.

Ecological risk assessment of persistent organic pollutants (POPs) in surface sediments from aquaculture system.

Organochlorinated pesticides (OCPs) and Polychlorinated biphenyls (PCBs) in the surface sediments from shrimp ponds in four regions of the northern part of the Central Java coast (namely Brebes, Tegal, Pemalang, and Pekalongan) were investigated. The highest concentration of ∑ OCPs was found in Brebes Regency, ranging from 68.1 ± 3.4 to 168.1 ± 9.8 µg kg-1 dw. As indicated by the DDT ratio and chlordane ratio, the value suggested that those compounds may mainly originate from historical inputs rather than a recent application. The concentrations of Æ© 7 indicator PCBs were determined, with the concentration ranged from 1.2 ± 0.7 µg kg-1 dw (Pekalongan) to 2.2 ± 0.4 µg kg-1 dw (Tegal). The most toxic PCB congener, PCB 118, was detected in all studied regions, with the highest proportion found in Tegal. Source analysis indicated that PCBs in the sediments mainly originated from Aroclor 1254 and Aroclor 1248. Compared to sediment quality guidelines (SQGs), some OCPs were found with concentrations which potentially posed an adverse effect. Our findings suggested that more attention should be paid to ensure sustainable shrimp culture facing such a risk of the OCPs and PCBs.

Assessment of the ecological and human health risks from metals in shrimp aquaculture environments in Central Java, Indonesia.

The occurrence and contamination level of seven important toxic metals (Cd, Cu, Co, Cr, Hg, Pb, and Zn) and three additional metals (Al, Fe, and Mn) in the water, sediment, and shrimp muscle in aquaculture areas located in Central Java, Indonesia, were investigated. The results suggest that the majority of metals have higher concentrations in the inlet followed by the outlet and ponds. Cd dissolved in the waters exhibited the highest level in Pekalongan (3.15 ± 0.33 µg L-1). Although Pb was not detected in the water, it was detected in the sediment, and the concentration ranged from 7.6 to 15.40 mg kg-1 dw. In general, the heavy metal concentrations in the sediments were found to decrease in the sequence Al > Fe > Mn > Zn > Cr > Cu > Co > Pb. Concentrations below the effects range low level based on the Canadian sediment quality guidelines were found for Cr, Cu, Pb, and Zn, whereas moderate sediment pollution (25-75 mg kg-1 dw) was observed for Cr (all regions), Cu (except in the Pekalongan region), and Zn (Brebes and Tegal regions) according to the US EPA standard. The status of the waters was evaluated by calculating a pollution index derived mostly from Mn and Zn. The ecological risk (geoaccumulation index (Igeo), contamination factor (CF), pollution load index (PLI), and potential ecological risk index (ERI)) determined in the sediments indicated that all studied areas had low to moderate contamination. The concentrations of all metals in shrimp were generally below the maximum limits for seafood, except for Zn (in all stations), Pb, and Cr (Tegal and Pekalongan). The hazard index values for metals indicated that consuming shrimp would not have adverse effects on human health.

A New Citrinin Derivative from the Indonesian Marine Sponge-Associated Fungus Penicillium citrinum.

Sponge-associated fungi are attractive targets for the isolation of bioactive natural products with different pharmaceutical purposes. In this investigation, 20 fungi were isolated from 10 different sponge specimens. One isolate, the fungus Penicillium citrinum strain WK-P9, showed activity against Bacillus subtilis JH642 when cultivated in malt extract medium. One new and three known citrinin derivatives were isolated from the extract of this fungus. The structures were elucidated by 1D and 2D NMR spectroscopy, as well as LC-HRMS. Their antibacterial activity against a set of common human pathogenic bacteria and fungi was tested. Compound 2 showed moderate activity against Mycobacterium smegmatis ATCC607 with a minimum inhibitory concentration (MIC) of 32 µg/mL. Compound 4 exhibited moderate growth inhibition against Bacillus subtilis JH642, B. megaterium DSM32, and M. smegmatis ATCC607 with MICs of 16, 16, and 32 µg/mL, respectively. Furthermore, weak activities of 64 µg/mL against B. subtilis DSM10 and S. aureus ATCC25923 were observed for compound 4.

Bioactivity of compounds secreted by symbiont bacteria of Nudibranchs from Indonesia.

The aims of this work are to isolate bacterial symbionts from nudibranchs and subsequently to determine anti-Methicillin resistant Staphylococcus aureus (MRSA), cytotoxicity and anti-Herpes simplex virus type 1 (HSV-1) activities of bio compounds. A total of 15 species of nudibranchs were collected from Karimunjawa and five species from Bali, respectively. A total of 245 bacteria isolates were obtained. The anti-MRSA activity screening activity indicated two active bacteria. Ethyl acetate extracts from supernatants, indicating extracelullar compounds, showed an inhibition zone against MRSA at concentrations of 500-1,000 µg/ml. DNA sequence analysis showed that the strain KJB-07 from Phyllidia coelestis was closely related to Pseudoalteromonas rubra, whereas the strain NP31-01 isolated from Phyllidia varicosa was closely related to Virgibacillus salarius. The extract of Pseudoalteromonas rubra was cytotoxic to Vero cells at a concentration of 75 µg/ml. The extract of V. salarius presented no cytotoxicity at concentrations of 5-1,000 µg/ml. No anti HSV-1 was observed for both isolated bacteria. This is the first study describing research on anti-MRSA, cytotoxicity and anti HSV-1 activity of bacterial symbionts from the viscera of nudibranch. Compounds produced by Pseudoalteromonas rubra and V. salarius, had potential anti-MRSA activity. However, only extracts from Pseudoalteromonas rubra showed cytotoxic effects on Vero cells. Three compounds were identified by LC/MS after purification from culture supernatant.

Comparative analysis of bacterial communities associated with healthy and diseased corals in the Indonesian sea.

Coral reef ecosystems are impacted by climate change and human activities, such as increasing coastal development, overfishing, sewage and other pollutant discharge, and consequent eutrophication, which triggers increasing incidents of diseases and deterioration of corals worldwide. In this study, bacterial communities associated with four species of corals: Acropora aspera, Acropora formosa, Cyphastrea sp., and Isopora sp. in the healthy and disease stages with different diseases were compared using tagged 16S rRNA sequencing. In total, 59 bacterial phyla, 190 orders, and 307 genera were assigned in coral metagenomes where Proteobacteria and Firmicutes were pre-dominated followed by Bacteroidetes together with Actinobacteria, Fusobacteria, and Lentisphaerae as minor taxa. Principal Coordinates Analysis (PCoA) showed separated clustering of bacterial diversity in healthy and infected groups for individual coral species. Fusibacter was found as the major bacterial genus across all corals. The lower number of Fusibacter was found in A. aspera infected with white band disease and Isopora sp. with white plaque disease, but marked increases of Vibrio and Acrobacter, respectively, were observed. This was in contrast to A. formosa infected by a black band and Cyphastrea sp. infected by yellow blotch diseases which showed an increasing abundance of Fusibacter but a decrease in WH1-8 bacteria. Overall, infection was shown to result in disturbance in the complexity and structure of the associated bacterial microbiomes which can be relevant to the pathogenicity of the microbes associated with infected corals.

Tunicate-associated bacteria show a great potential for the discovery of antimicrobial compounds.

Tunicates (Ascidians, sea squirts) are marine protochordates, which live sedentary or sessile in colonial or solitary forms. These invertebrates have to protect themselves against predators and invaders. A most successful strategy, to not being eaten by predators and prevent pathogenic microorganisms to settle, is the usage of chemical molecules for defence. To accomplish this, tunicates take advantage of the specialized metabolites produced by the bacteria associated with them. Therefore, the microbiome of the tunicates can be regarded as a promising bioresource for bacterial strains producing compounds with antibacterial activity. The aim of this study was to test this hypothesis by (i) isolation of tunicate-associated bacteria, (ii) analysis of the antibacterial activities of these strains, and (iii) purification and structure elucidation of an active compound derived from this bioresource. In total, 435 bacterial strains were isolated and thereof 71 (16%) showed antibacterial activity against multidrug resistant (MDR) bacteria. Therefrom, the ethyl acetate crude extracts from liquid fermentations of 25 strains showed activity against MDR Extended-Spectrum Beta-Lactamase (MDR-ESBL) Escherichia coli, MDR Bacillus cereus, Micrococcus luteus, and Bacillus megaterium. Phenotypic analysis based on 16S rDNA sequencing revealed the active strains belonging to different genera and phyla, like Bacillus, Pantoea, Pseudoalteromonas, Salinicola, Streptomyces, Vibrio and Virgibacillus. To obtain first insights into the molecules responsible for the antibacterial activities observed, strain Pseudoalteromonas rubra TKJD 22 was selected for large-scale fermentation and the active compound was isolated. This allowed the purification and structure elucidation of isatin, a compound known for its strong biological effects, thereunder inhibition of Gram-positive and Gram-negative pathogens.

Two new aromatic polyketides from a sponge-derived Fusarium.

In our natural product screening program from marine fungi, two new aromatic polyketides karimunones A (1) and B (2) and five known compounds (3-7) were isolated from sponge-associated Fusarium sp. KJMT.FP.4.3 which was collected from an Indonesian sponge Xestospongia sp. The structures of these compounds were determined by the analysis of NMR and MS spectroscopic data. The NMR assignment of 1 was assisted by DFT-based theoretical chemical shift calculation. Compound 2 showed antibacterial activity against multidrug resistant Salmonella enterica ser. Typhi with a MIC of 125 µg/mL while 1 was not active.

Organophosphate pesticide concentrations in coral tissues of Indonesian coastal waters.

In this study we evaluated the persistence of diazinon, chlorpyrifos, profenofos, parathion, malathion and ethion in dead coral tissues of Indonesian coastal waters (Java, Bali, Sulawesi and Komodo). Comparison of the residue levels in coral tissues showed that the highest presence of organophosphate concentrations was detected in a coral sample collected from Java coastal waters. While medium amounts of a contaminant diazinon can still lead to detectable in Bali and Sulawesi coastal waters. Prominent contamination of organophosphate was not found in a sample collected from Komodo. Neither parathion nor malathion were detected in any of the samples. This result implies that the geographical variations of organophosphate compounds are determined by the possible usage of these chemicals around coastal waters at the present or in the past. There is need for further work to identify sources and fate of pesticide contaminants, as well as to improve monitoring of pesticide use.