{"id":62920,"date":"2024-12-30T11:20:48","date_gmt":"2024-12-30T11:20:48","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=62920"},"modified":"2025-01-06T18:27:28","modified_gmt":"2025-01-06T18:27:28","slug":"antibacterial-potential-of-pseudomonas-aeruginosa-isp1rl4-isolated-from-seaweed-eucheuma-cottonii-against-multidrug-resistant-bacteria","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no4\/antibacterial-potential-of-pseudomonas-aeruginosa-isp1rl4-isolated-from-seaweed-eucheuma-cottonii-against-multidrug-resistant-bacteria\/","title":{"rendered":"Antibacterial Potential of Pseudomonas aeruginosa ISP1RL4 Isolated from Seaweed Eucheuma cottonii Against Multidrug-resistant Bacteria"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Bacteria that are resistant to multiple drugs (MDR)\nhave become a significant public health issue due to their increasing\nprevalence1<\/sup>. The excessive and improper use of\nantibiotics has driven the development of resistance in various bacteria,\nleading to the accumulation of multiple resistance gene2<\/sup>s. A number of Gram-positive and negative\nbacterial species have transformed into resistance strains against different\ntypes of antibiotics such as \u03b2-lactamase classes and vancomycin3<\/sup>. Furthermore, the emergence of broad-spectrum\nbeta-lactamase (ESBL) enzymes in Enterobacteriaceae has worsened the global\nantibiotic resistance crisis4<\/sup>. Although raising public awareness to\nuse antibiotic rationally remains essential, the urgent need for new and potent\nantibiotic producers to combat MDR bacteria cannot be overstated.<\/p>\n\n\n\n

For decades, terrestrial microorganisms particularly\nbacteria and fungi have been the primary target discovery for novel\nantibacterial compounds5<\/sup>. Nevertheless, many of these antibiotic\ncompounds have been previously isolated and reported, leading to a reduction in\nthe novelty rate of these compounds6<\/sup>. Conversely, marine habitats offer\nvariety of bioactive molecules, including antibiotics that hold significant\npharmaceutical importance7<\/sup>. Various marine species have been\nproven to produce arrays of bioactive molecules, with pharmaceutical potential\nincluding that of antibiotic compounds8<\/sup>. However, to perform clinical test and\nto synthesize specific bioactive molecules require a large number of biomasses\nto acquire sufficient extracts9<\/sup>. Furthermore, ecological concerns\nprevent the direct cultivation of such substantial biomass in nature. As a\nresult, the focus has shifted towards bioprospecting marine bacteria,\nparticularly those associated with marine organisms, as they offer faster and\nrelatively easier cultivability under laboratory conditions, making them\nsuitable for synthesizing compounds of interest10,11<\/sup>. <\/p>\n\n\n\n

Marine microorganisms, particularly bacteria, have\nemerged as significant sources of novel antibacterial compounds. This is\nlargely due to their unique evolutionary adaptations to the marine environment,\nwhich have equipped them with diverse biochemical pathways for producing\nbioactive metabolites12\u201314<\/sup>. Among these microorganisms,\nmembers of the genus Bacillus are particularly noteworthy. They are known to\nsynthesize a variety of antimicrobial substances, including polyketides,\nlipopeptides, and bacteriocins, which exhibit broad-spectrum antimicrobial\nactivity against various pathogens15\u201317<\/sup>. Other marine bacterial species\nsuch as actinobacteria are among the prominent producers of antibacterial\ncompounds, showcasing their potential in drug discovery and development18<\/sup>. Overall, marine bacteria represent a\nrich reservoir of antibacterial compounds with diverse mechanisms of action.<\/p>\n\n\n\n

Eucheuma cottonii,<\/em> a\nseaweed species commonly found in Indonesia, is rich in nutrients and valuable\ncompounds like carrageenan, flavonoids, and tannins19,20<\/sup>. Like other seaweeds, it forms a\nbeneficial partnership with bacteria, which help it grows, develops, and\ndefends against threats by producing antibacterial substances21<\/sup>. However, research on E. cottonii<\/em>-associated\nbacteria is rather scarce22,23<\/sup>. In a previous study, Aeromonas<\/em>\nbacteria isolated from E. cottonii<\/em>, actively inhibited Staphylococcus\naureus <\/em>and Escherichia coli<\/em>22<\/sup>. Additionally, twenty-three bacterial\nisolates were discovered on E. cottonii<\/em> in a recent study conducted in\nthe coastal waters of Buleleng, Bali, with six of these isolates exhibiting\nantibacterial activity against S. aureus<\/em>, Streptococcus mutans<\/em>, E.\ncoli<\/em>, and Klebsiella pneumoniae<\/em>23<\/sup>.<\/em>  <\/p>\n\n\n\n

Among six reported\npotential isolates, a bacterial isolate encoded as ISP1RL4 specifically\ndisplayed the strongest antibacterial activity23<\/sup>. However, it is remained unclear to\nwhat species this isolate is assigned. Therefore, it requires further molecular\nidentification and characterization. In addition, it is intriguing to evaluate\nantibacterial activity of the isolate crude extract against multidrug-resistant\nbacteria to confirm its previous reported antibacterial potential based on agar\nblock method23<\/sup>. Furthermore, chemical profiling on the\nbacterial extract was performed to provide a clear insight on the possible\npromising antibacterial compounds. <\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Materials <\/strong><\/p>\n\n\n\n

The ISP1RL4 bacterium was isolated from the seaweed E.\ncottonii<\/em> from Patas village, Buleleng Regency, Bali, Indonesia23<\/sup>. The pure culture was grown in slant\nagar and stored at 4o<\/sup>C until further used. Gram staining and\nZiehls-Neelsen staining kits were purchased from local suppliers. The test\npathogenic bacteria used were Methicilin-resistant\nStaphylococcus aureus<\/em> (MRSA), Escherichia coli<\/em> Extended-spectrum\nbeta-lactamases (ESBL), Klebsiella pneumoniae<\/em> ESBLand\nESBL Acinetobacter baumanii<\/em> were used as previously described24<\/sup>. All other analytical grade chemicals\nsuch as aqua distillation, ethyl acetate was purchased from local suppliers. <\/p>\n\n\n\n

DNA isolation<\/strong><\/p>\n\n\n\n

Cell mass of ISP1RL4 pure culture was grown in 1.5 mL\nsterile International Streptomyces<\/em> Project-2 (ISP-2) broth medium and\nwas incubated at 28o<\/sup>C for 7 days. Bacterial DNA was extracted using\na bacteria DNA preparation kit following the protocol (Jena Bioscience,\nGermany). DNA concentration was determined by a Nanodrop with a 260\/280 nm\nratio25<\/sup>. <\/p>\n\n\n\n

16SrDNA gene\namplification and phylogenetic analysis <\/strong><\/p>\n\n\n\n

Molecular identification was performed by PCR\namplifying 16S rRNA gene using two primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and\nreverse primer 1492R (5′-CGGTTACCTTGTTACGACTT-3′) based on the previously\ndescribed protocol24<\/sup>. PCR product was sent for Sanger\nsequencing to PT Genetika Science, Tangerang, Indonesia\n(https:\/\/ptgenetika.com\/). The quality of raw sequence was checked using sequence\nscanner V.2.0 and low-quality sequences were trimmed using ChromasPro 2.1.10.\nFinally, sequences were assembled using software DNAMAN Ver.9.0. The nucleotide\nsequence was compared against a database of known sequences using the n-BLAST\nmethod on the NCBI BLAST platform (https:\/\/blast.ncbi.nlm.nih.gov\/Blast.cgi).\nFollowing the BLAST search, the ten most relevant sequences were chosen and\ntheir base information was used to construct a phylogenetic tree. The\nNeighbor-joining method with the Kimura-2 parameter model was implemented in\nMEGA X software (https:\/\/www.megasoftware.net)24<\/sup>. The reliability of the tree was\nassessed using bootstrap analysis with 1000 repetitions. <\/p>\n\n\n\n

Scanning Electron Microscope (SEM) preparation<\/strong><\/p>\n\n\n\n

The SEM observation method was adapted from a\nprevious study26<\/sup>.Cell mass of ISP1RL4 isolate\nwere obtained from overnight culture on ISP-2 medium. Cell biomass was washed\nby Phosphate Buffer Saline (Merck, Germany) 1x. The cell biomass was soaked\nwith 2% glutaraldehyde in PBS for 2 h at room temperature. The cell biomass was\nseparated from solution was centrifugation method (13,000 rpm for 5 min). The\ncell biomass was rinsed with PBS twice. \nThe sodium tetraoxide (1% in PBS) was added in cell biomass and was\nsoaked for 2 h in room temperature. Sodium tetraoxide was removed by\ncentrifugation method. Before coating process, the cell biomass was dehydrated\nwith serial ethanolic solution (70% for 10 min, 96% for 10 min, absolute\nethanol for 10 min) and followed by centrifugation method to obtain ethanolic\nfree cell biomass. The cell biomass was placed on carbon tape and subsequently\napplying a gold coating through a sputtering process. The scanning electron\nmicroscope was set to a high vacuum, 5 kV accelerating voltage, 30% spot\nintensity, and magnifications of 2,000x, 5,000x, and 10,000x (Hitachi SU3500,\nJapan)26<\/sup>.<\/p>\n\n\n\n

Fermentation and Extraction Condition<\/strong><\/em><\/p>\n\n\n\n

Ten mL of ISP1RL4 pre-cultured in ISP-2 media was\nadded aseptically into 90 mL sterile ISP-2 broth and the mixture was fermented\nin 14 days at 150 rpm27<\/sup>. After reaching the fermentation\nperiod, the supernatant was separated from cell mass using Whatman paper no\n1.  Subsequently, it was extracted using\n100 mL of ethyl acetate pro Analisa (Merck, Germany). A separatory funnel was\nused to separate the organic layer from liquid layer. The extraction and\nseparation process were repeated two times. Finally, the pool of organic layers\nwas evaporated in a vacuum evaporator to achieve the final extract for further\nantimicrobial screening28<\/sup>. <\/p>\n\n\n\n

Antibacterial Assay of Against Selected\nMultidrug-Resistant Bacteria<\/strong><\/p>\n\n\n\n

Screening of the antibacterial activity of ISP1RL4\nisolate crude extract was performed following disc diffusion assays. Thirty\nmicroliters of ISP1RL4 crude extract were applied to sterile 6-millimeter\ndiameter papers disc. These discs were then placed in triplicates on LB agar\nplates containing various multidrug-resistant bacteria, including MRSA,\nextended-spectrum beta-lactamase (ESBL) Escherichia coli<\/em> and ESBL Klebsiella\npneumoniae<\/em>, and ESBL Acinetobacter baumannii<\/em>. These plates were stored\nat a 37o<\/sup>C incubator for one day. The diameter of inhibition zone\nformed against each bacterial target was quantify using a digital caliper.<\/p>\n\n\n\n

Thin Layer Chromatography and Antibacterial Assay <\/strong><\/p>\n\n\n\n

The extract was separated and purified using thin\nlayer chromatography (TLC). Briefly, TLC of the substance was applied on the GF-254\nsilica gel and developed in a solvent system containing n-hexane and ethyl\nacetate in a ratio of 4:6. Distinct components were identified by viewing them\nunder ultraviolet light (254 nm), subsequently their retention factors (Rf\nvalues) were determined. Each visible spot on the TLC plate was then collected,\ndissolved in the same solvent mixture, and tested for antibacterial activity\nagainst the MDR bacterial test using the Kirby-Bauer method as previously\ndescribed.<\/p>\n\n\n\n

Gas Chromatography\/Mass Spectrometry preparation\nand analysis  <\/strong><\/p>\n\n\n\n

Volatile compounds that constitute ethyl acetate\ncrude extract of ISP1RL4. Briefly, 0.1 gram of the crude extract was prepared\nand sent to the Forensic Laboratory Polda Bali, Indonesia for further analysis.\nThe extract was injected to the GC\/MS instrument (Agilent Technologies 7890B\/Agilent\nTechnologies 5977B) based on the following setting: HP-5ms ultra inert column\n30 m x250 \u00b5m x 0.25 \u00b5m, oven temperature (-60o<\/sup>C to 325o<\/sup>C),\nmode (splitless), pressure (25.523 psi), total flow (20.9 ml\/min), average\nvelocity (62.662 cm\/sec), purge flow to split vent (15 mL\/min at 0.75 min), and\ngas (He). Chromatograms were analyzed by matching the compound fragments from\neach chromatogram peak with literature to determine the type of content and to\nunderstand the biological properties of the discovered compounds.<\/p>\n\n\n\n

Liquid Chromatography High Resolution Mass\nSpectrometry (LC-HRMS) preparation and analysis<\/strong><\/p>\n\n\n\n

Metabolomic analysis of ISP1RL4 ethyl acetate was\nperformed in Ultra-High-Performance Liquid Chromatography coupled to untargeted\nHigh-Resolution Mass Spectrometry (Thermo Scientific Dionex Ultimate 3000 RSLC\nNano UHPLC paired with Thermo Scientific Q Extractive (Thermo Fisher\nScientific, Massachusetts, USA). The machine was run by following previously\ndescribed procedure26<\/sup>. <\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

Morphological Observation<\/strong><\/p>\n\n\n\n

The cell morphology of\nthe ISP1RL4 bacterial isolate, as observed under a microscope after Gram\nstaining, categorizes the isolate as a Gram-negative bacterium with\nbacilli-shaped cells. (Figure 1A). This observation was confirmed by examining\nbacterial cells of the ISP1RL4 isolate under a scanning electron microscope,\nwhich provided a clearer appearance with smooth surface, approximately 5 \u00b5m in\nlength, and exhibiting attachment to each other (Figure 1B). On agar plate, the\nISP1RL4 pure isolate had colonies with irregular surface shapes, firmly\nattached to the media, with a powdery consistency, and rough textured and dull\nsurfaces, and had a grayish-yellow pigmentation with the reverse of colony\ncolor with light yellow green pigmentation on ISP-2 agar at 11 days of age\n(Figure 1C). Based on the results of Ziehl-Neelsen’s acid-resistant staining, ISP1RL4 isolate was a non-acid fast bacterium because it\ncould not retain the red dye from carbolic fuchsin after being dripped with\nalcoholic acid. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Microscopic view of ISP1RL4 isolate bacterial cells (A) by Gram staining using 1000x magnification and (B) by Scanning electron microscopy using 10,000x magnification and (C) macroscopic view of ISP1RL4 pure isolate on agar plate.<\/strong><\/p>\n<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Molecular Identification <\/strong><\/p>\n\n\n\n

DNA isolation of ISP1RL4 exhibited concentration of\n296 ng\/\u00b5L with a DNA purity level of 1.78 nm at a ratio of A260\/280 nm.  Based on the result of the alignment of\nISP1RL4 isolate, it was revealed that ISP1RL4 isolate had DNA sequences with\n100% homology (percentage identity) with Pseudomonas aeruginosa <\/em>strain\nM4. The PCR results of the 16S rRNA product of ISP1RL4 has a DNA sequence of\nless than 1500 bp, which is 1411 bp. Percentage identity or what is known as\nthe homology value is a percentage that indicates how well the input DNA\nsequence matches the target DNA sequence. The results of the phylogenetic tree\nconstruction as shown in Figure 2 showed that the ISP1RL4 isolate was located\nin the same clade and branch, and shared the same node with Pseudomonas\naeruginosa <\/em>strain M4 on the phylogenetic tree. According to Figure 3, the\nphylogenetic tree of ISP1RL4 isolate formed was not a paraphyletic group of\nphylogenetic trees, but a monophyletic. Based on molecular identification,\nISP1RL4 isolate can be identified as Pseudomonas aeruginosa<\/em> sp. and\nphylogenetically related to Pseudomonas aeruginosa <\/em>strain M4. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: ISP1RL4 isolate phylogenetic tree which describes the phylogenetic position of ISPIRL4 isolate with other bacteria in one clade or another clade (Staphylococcus hominis <\/em>strain DM 122 as the outer group).<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Evaluation\nof Antibacterial Activity of ISP1RL4 Crude Extract Against Multidrug-resistant\nBacteria<\/strong><\/p>\n\n\n\n

The results showed that ISP1RL4 isolate could inhibit the growth of\nthree multidrug-resistant bacteria, except for A.\nbaumanii <\/em>(Table 1). ISP1RL4 extract had moderate\ncategory of antibacterial activity with an average inhibition zone of >9 mm.\nThe highest antibacterial activity of ISP1RL4 extract was shown in multidrug\nresistant E. coli<\/em> ESBL bacteria with an inhibition zone diameter of 10.3 \u00b1 3.0 mm.\nISP1RL4 extract had the lowest ability to inhibit the activity of MRSA bacteria\nwith diameter zone of inhibition of 9.0 \u00b1 1.0 mm. <\/p>\n\n\n\n

Ethyl acetate extract of ISP1RL4 isolate was able to inhibit the growth\nof the Gram-positive and Gram-negative multidrug resistant bacteria as shown in\nFigure 3. Ethyl acetate crude extract of ISP1RL4 was more effective to inhibit\nthe growth of Gram-negative multidrug resistant bacteria especially E. coli <\/em>ESBL\nas shown in Table 1 compared to MRSA. <\/p>\n\n\n\n

Table 1: Antibacterial activity of the crude extract of ISP1RL4 isolate against multi-drug resistant bacteria<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n
\n

Multidrug resistant bacteria<\/strong><\/p>\n<\/td>\n

\n

Zone of Inhibition (mm)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

MRSA<\/p>\n<\/td>\n

\n

9,0 \u00b1 1,0<\/p>\n<\/td>\n<\/tr>\n

\n

E. coli <\/em>ESBL<\/p>\n<\/td>\n

\n

10,3 \u00b1 2,0<\/p>\n<\/td>\n<\/tr>\n

\n

K. pneumoniae <\/em>ESBL<\/p>\n<\/td>\n

\n

9,4 \u00b1 0,1<\/p>\n<\/td>\n<\/tr>\n

\n

A. baumanii<\/em><\/p>\n<\/td>\n

\n

0\u00b1 0,1<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Notes: Average diameter of the inhibition zone for each isolate was measured from three replications.<\/p>\n\n\n\n
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Figure 3: Antibacterial activity of ISP1RL4 crude extract against multidrug-resistant bacteria (A) Methicillin-resistant Staphylococcus aureus <\/em>(MRSA), <\/em>(B) Klebsiella pneumoniae <\/em>ESBL<\/strong><\/p>\n<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Fraction Analysis of Active Compound Extract Filtrate in ISP1RL4\nIsolate<\/strong><\/p>\n\n\n\n

The results of TLC\nanalysis (thin layer chromatography) revealed that there was only one spot\n(Figure 4) that appeared on the GF254<\/sub> TLC plate (Table 2). Fraction\nF1 had an Rf<\/em> value of 0,06. The fraction was tested for antibacterial\nactivity on the test bacteria. The highest inhibition of the fraction was\nobserved against MRSA (Figure 5).<\/p>\n\n\n\n

Table 2: Antibacterial activity of the active compound fraction of ISP1RL4 isolate on the test bacteria<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
\n

Fraction<\/strong><\/p>\n<\/td>\n

\n

Rf <\/em>value<\/strong><\/p>\n<\/td>\n

\n

<\/a><\/a><\/a><\/a>Zone of Inhibition (mm)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

MRSA<\/strong><\/p>\n<\/td>\n

\n

E. coli <\/em>ESBL<\/strong><\/p>\n<\/td>\n

\n

K. pneumoniae <\/em>ESBL<\/strong><\/p>\n<\/td>\n

\n

A. baumanii<\/em><\/strong> ESBL<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/strong><\/p>\n<\/td>\n

\n

0,06<\/p>\n<\/td>\n

\n

18\u00b14<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

7,4\u00b10,2<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

K+ (Nalidixic acid <\/em>30 \u00b5g)<\/strong><\/p>\n<\/td>\n

\n

9\u00b10,3<\/p>\n<\/td>\n

\n

9,8\u00b10,2<\/p>\n<\/td>\n

\n

9,2\u00b10,2<\/p>\n<\/td>\n

\n

9,5\u00b10,3<\/p>\n<\/td>\n<\/tr>\n

\n

K- (Ethyl acetate)<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Notes: K+: Positive control; K-: Negative control. The average diameter of the inhibition zone for each isolate was measured from three replications<\/p>\n\n\n\n
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Figure 4: Spot visualization of ISP1RL4 isolate  extract fraction on GF254<\/sub> silica gel plate<\/strong><\/p>\n<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Notes: Yellow circle: spot on the\nfraction that had successfully appeared.<\/p>\n\n\n\n\n\n
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Figure 5: Diameter zone of inhibition of the ethyl acetate fraction against MRSA<\/p>\n<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

LC-HRMS Analysis of Active Compound in\nISP1RL4 Isolate<\/strong><\/p>\n\n\n\n

The results of LC-HRMS analysis of the crude ethyl acetate extract\nisolate ISP1RL4 detected 381 compounds with different peak percentages and\nretention times which were successfully identified in the LC-HRMS chromatogram\nshown in Figure 6. Eleven antibacterial compounds detected in the ethyl acetate\ncrude extract of ISP1RL4 isolate based on LC-HRMS could be seen in Table 3. The\nhighest detected compound was 2-Amino-1,3,4-octadecanetriol (11,21 % relative\nabundance). The GC-MS analysis of the crude ethyl acetate isolate ISP1RL4\ndetected 11 antibacterial compounds as presented in Table 4. <\/strong><\/p>\n\n\n\n\n\n
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Figure 6: LC-HRMS chromatogram of the ethyl acetate extract of ISP1LR4 isolate<\/strong><\/p>\n<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 3: Antibacterial compounds detected in the ethyl acetate crude extract of ISP1RL4 isolate based on LC-HRMS results<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Name<\/strong><\/p>\n<\/td>\n

\n

Formula<\/strong><\/p>\n<\/td>\n

\n

Cal. Molecular Weight<\/strong><\/p>\n<\/td>\n

\n

Compound characteristic<\/strong><\/p>\n<\/td>\n

\n

Activity<\/strong><\/p>\n<\/td>\n

\n

% relative abundance<\/strong><\/p>\n<\/td>\n

\n

Reference<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

2-Amino-1,3,4-octadecanetriol<\/p>\n<\/td>\n

\n

C18<\/sub>H39<\/sub>NO3<\/sub><\/p>\n<\/td>\n

\n

317.29191<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

11.21<\/p>\n<\/td>\n

\n

29<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Bis(4-ethylbenzylidene) sorbitol<\/p>\n<\/td>\n

\n

C24<\/sub>H30<\/sub>O6<\/sub><\/p>\n<\/td>\n

\n

414.20368<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

4.81<\/p>\n<\/td>\n

\n

30<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Dextromethorphan<\/p>\n<\/td>\n

\n

C18<\/sub>H25<\/sub>NO<\/p>\n<\/td>\n

\n

271.19315<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

4.53<\/p>\n<\/td>\n

\n

31<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Dibutyl phthalate<\/p>\n<\/td>\n

\n

C16<\/sub>H22<\/sub>O4<\/sub><\/p>\n<\/td>\n

\n

278.15142<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

4.41<\/p>\n<\/td>\n

\n

32<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

C14-Dihydroceramide<\/p>\n<\/td>\n

\n

C32<\/sub>H65<\/sub>NO3<\/sub><\/p>\n<\/td>\n

\n

511.49607<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

4.17<\/p>\n<\/td>\n

\n

33<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

C16-Dihydroceramide<\/p>\n<\/td>\n

\n

C34<\/sub>H69<\/sub>NO3<\/sub><\/p>\n<\/td>\n

\n

539.52751<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

2.76<\/p>\n<\/td>\n

\n

33<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

6-Methoxyquinoline<\/p>\n<\/td>\n

\n

C10<\/sub>H9<\/sub>NO<\/p>\n<\/td>\n

\n

159.06818<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

2.10<\/p>\n<\/td>\n

\n

34<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

D-(+)-Maltose<\/p>\n<\/td>\n

\n

C12<\/sub>H22<\/sub>O11<\/sub><\/p>\n<\/td>\n

\n

342.11637<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

1.79<\/p>\n<\/td>\n

\n

35<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Propranolol<\/p>\n<\/td>\n

\n

C16<\/sub>H21<\/sub>NO2<\/sub><\/p>\n<\/td>\n

\n

259.15684<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

1.63<\/p>\n<\/td>\n

\n

36<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

1-Stearoylglycerol<\/p>\n<\/td>\n

\n

C21<\/sub>H42<\/sub>O4<\/sub><\/p>\n<\/td>\n

\n

358.30770<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

1.52<\/p>\n<\/td>\n

\n

37<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Armillaramide<\/p>\n<\/td>\n

\n

C34<\/sub>H69<\/sub>NO4<\/sub><\/p>\n<\/td>\n

\n

555.52202<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

1.12<\/p>\n<\/td>\n

\n

33<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 4: Antibacterial compounds detected in the ethyl acetate crude extract of ISP1RL4 isolate based on GC-MS results<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Name<\/strong><\/p>\n<\/td>\n

\n

Formula<\/strong><\/p>\n<\/td>\n

\n

Molecular Weight (g\/mol)<\/strong><\/p>\n<\/td>\n

\n

Compound characteristic<\/strong><\/p>\n<\/td>\n

\n

Activity<\/strong><\/p>\n<\/td>\n

\n

% relative abundance<\/strong><\/p>\n<\/td>\n

\n

Reference<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

2-Hexanol<\/p>\n<\/td>\n

\n

C6<\/sub>H14<\/sub>O<\/p>\n<\/td>\n

\n

102.17<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

10.94<\/p>\n<\/td>\n

\n

38<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

3-Hexanol<\/p>\n<\/td>\n

\n

C6<\/sub>H14<\/sub>O<\/p>\n<\/td>\n

\n

102.17<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

9.86<\/p>\n<\/td>\n

\n

39<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

3-Pentanol, 2-methyl-<\/p>\n<\/td>\n

\n

C6<\/sub>H14<\/sub>O<\/p>\n<\/td>\n

\n

102.17<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

9.86<\/p>\n<\/td>\n

\n

40<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

2-Hexanone<\/p>\n<\/td>\n

\n

C6<\/sub>H12<\/sub>O<\/p>\n<\/td>\n

\n

100.16<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

8.60<\/p>\n<\/td>\n

\n

41<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Isobutyl acetate<\/p>\n<\/td>\n

\n

C6<\/sub>H12<\/sub>O2<\/sub><\/p>\n<\/td>\n

\n

116.16<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

7.62<\/p>\n<\/td>\n

\n

42<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Cyclotrisiloxane, hexamethyl-<\/p>\n<\/td>\n

\n

C12<\/sub>H22<\/sub>O11<\/sub><\/p>\n<\/td>\n

\n

222.46<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

7.12<\/p>\n<\/td>\n

\n

43<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

3-Hexanone<\/p>\n<\/td>\n

\n

C18<\/sub>H30<\/sub>O3<\/sub>S<\/p>\n<\/td>\n

\n

100.16<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

4.56<\/p>\n<\/td>\n

\n

44<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Toluene<\/p>\n<\/td>\n

\n

C6<\/sub>H5<\/sub>CH3<\/sub><\/p>\n<\/td>\n

\n

92.14<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

3.81<\/p>\n<\/td>\n

\n

45<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Ethylbenzene<\/p>\n<\/td>\n

\n

C8<\/sub>H10<\/sub><\/p>\n<\/td>\n

\n

106.16<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

2.40<\/p>\n<\/td>\n

\n

46,47<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

o-xylene<\/p>\n<\/td>\n

\n

C8<\/sub>H10<\/sub><\/p>\n<\/td>\n

\n

106.16<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

2.40<\/p>\n<\/td>\n

\n

48\u201350<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Actinobolin<\/p>\n<\/td>\n

\n

C13<\/sub>H20<\/sub>N2<\/sub>O6<\/sub><\/p>\n<\/td>\n

\n

135.21<\/p>\n<\/td>\n

\n

Organic compound<\/p>\n<\/td>\n

\n

Antibacterial<\/p>\n<\/td>\n

\n

1.10<\/p>\n<\/td>\n

\n

51<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

The study investigated the antibacterial properties\nof a bacterial strain, ISP1RL4, isolated from E. cottonii<\/em> seaweed. Microscopic\nanalysis revealed that the isolate was rod-shaped and Gram-negative. Molecular\nidentification confirmed that the isolate ISP1RL4 identified as Pseudomonas\naeruginosa<\/em>. The isolation of the bacterium from seaweed aligns with the\nknown distribution of Pseudomonas species, which are frequently found in marine\necosystems52,53<\/sup>. Marine P. aeruginosa<\/em> has\nbeen a subject of interest due to its diverse characteristics and potential\napplications. Studies have highlighted the adaptability of P. aeruginosa<\/em>\nin various environments, including marine habitats, where it demonstrates\nunique patterns of cultivability and survival, indicating physiological\nadaptations to oceanic conditions54<\/sup>.<\/p>\n\n\n\n

Our study has provided further evidence to support the antibacterial potential of P. aeruginosa <\/em>ISP1RL4, as initially suggested by block agar experiments23<\/sup>. The ethyl acetate extract derived from P. aeruginosa<\/em> ISP1RL4 effectively suppressed the growth of both Gram-positive and Gram-negative MDR bacteria. Additionally, P. aeruginosa<\/em> has been identified as a source of antimicrobial activity against multidrug-resistant pathogens, with certain marine isolates demonstrating efficacy against bacteria like S. aureus55<\/sup>.<\/em> The production of antimicrobial metabolites by marine Pseudomonas strains further underscores their potential as sources of novel antibacterial agents. The antibacterial properties exhibited by the ethyl acetate extract suggest that its components have a wide range of antibacterial activity56<\/sup>. This result aligns with prior research highlighting the broad-spectrum antibacterial properties of P. aeruginosa<\/em> against various bacterial species. In terms of the degree of inhibition, the extract exhibited moderate to strong antibacterial activity, with inhibition zones ranging from 9.8 to 11 millimeters57<\/sup>. Notably, the purified extract effectively suppressing MRSA bacterial target compared to that of other test bacteria. This finding highlights the selective antibacterial activity of the purified extract, demonstrating a particular efficacy against MRSA compared to other bacterial strain. In addition, discrepancy diameter zone of inhibition could be due to the absence of an outer membrane with lipopolysaccharide in Gram-positive bacteria, making them more susceptible to antibacterial compounds compared to Gram-negative bacteria58,59<\/sup>. The produced diameter zone of inhibition may vary which can be attributed to the different types of secondary metabolites produced, different chemical composition, concentration, and polarity. Additionally, the morphological and physiological characteristics of each bacterial strain influence these results60<\/sup>. <\/p>\n\n\n\n

Variations in the type of active compound content\nwere found in the LC-HRMS and GC-MS results. Active compounds were found to be\nmore diverse and numerous in LC-MS results than GC-MS. The ability of LC-HRMS\nto generate exact mass measurements and molecular formulas for unknown\ncompounds in an extract could contribute to this observation. Additionally, this\nmethod excels at determining chemical structures with high sensitivity, even\nwhen working with small sample sizes and limited time61,62<\/sup>. Comparing to GC-MS, a commonly\nemployed technique for characterizing chemotypes from a sample, the instrument\nwas limited to analyzing non-polar and\/or volatile compounds63<\/sup>. This had covered a broader spectrum\nof compounds found in LC-HRMS which had been shown by the identification of\nnon-volatile compounds such as sorbitol and ceramides.<\/p>\n\n\n\n

The active compound of P. aeruginosa <\/em>ISP1RL4\nethyl acetate extract found in LC-HRMS was mainly dominated by\n2-Amino-1,3,4-octadecanetriol (11.2%), which belong to the phytosphingosine\ncompound class. Phytosphingosine is a long-chain sphingolipid base consisting\nan amino alcohol with 18 carbon atoms typical in plants, which has\nantibacterial properties. Phytosphingosine, at a concentration of 15.9 \u03bcg\/mL,\neffectively killed 95% of the three bacterial species: P. syringae<\/em> pv.\ntomato, A. tumefaciens<\/em>, and R. radiobacter29<\/sup><\/em>.\nC14-Dihydroceramide, C16-Dihydroceramide, and Armillaramide are ceramides\nreported for the first time that had been isolated from Eucheuma cottonii<\/em>-associated\nbacterial. A study discovered that short-chain ceramides and a\n\u03c9-azido-C6-ceramide exhibited antibacterial activity towards Neisseria\nmeningitidis <\/em>and N.<\/em> gonorrhoeae<\/em>33<\/sup>.Ceramides found in Cissus\nincisa<\/em> leaves had an antibacterial activity against nine\nmultidrug-resistant bacteria, with the most significant inhibition observed\nagainst Gram-negative bacteria, particularly carbapenem-resistant Acinetobacter\nbaumannii<\/em> at a concentration of 50 \u03bcg\/mL64<\/sup>. \nBased on LC-HRMS results, a sorbitol compound was found in the ethyl\nacetate extract of P. aeruginosa <\/em>ISP1RL4, namely Bis(4-ethylbenzylidene)\nsorbitol, which had also been found in black cumin extract with the best\nantibacterial activity against MRSA via in silico methods30<\/sup>. <\/p>\n\n\n\n

Despite the confirmed antibacterial potential of\nethyl acetate extract of P. aeruginosa ISP1RL4, there are a number of\nlimitations that need to be improved for future studies. Firstly, the observed\nactivity resulted from crude extracts therefore an optimization is required\nsuch as performing NMR analysis to determine the exact active compounds65<\/sup>. Secondly, the fermentation volume at\nthe current study was set at 100 mL which resulted in a low yield of an active\nextract, therefore a higher volume e.g. 1 liter is essential to obtain more\nextracts. Thirdly, the current screening was mainly focused on measuring the\ndiameter zone of inhibition, however no information is available on the median\nlethal dose (LD50) of the extract which is crucial for a more comprehensive\nantibacterial analysis. Fourthly, more MDR bacterial strains that are among\nhuman pathogens such as Enterobacterium faecium<\/em>, Enterobacterium faecalis<\/em>,\nand Streptococcus pneumoniae<\/em>1<\/sup> need to be included to evaluate\nantibacterial spectrum of the ethyl acetate extract of P. aeruginosa<\/em>\nISP1RL4. Lastly, the current study has not evaluated toxicity of the compounds,\nso the future study should be focused to screen for in vitro toxicity test such\nas MTT assays and lactate dehydrogenase. Such screening will ensure safety and\nefficacy of the extract.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

In conclusion, this study has confirmed\nantibacterial potential of the isolate Pseudomonas aeruginosa<\/em> ISP1RL4\nagainst selected multidrug-resistant bacteria. Analysis of chemical profiles of\nthe crude extract has identified a number of promising antibacterial molecules\nthat potentially can be synthesized by P. aeruginosa<\/em> ISP1RL4. Future\nstudies would be focused to improve some limitations of the current results\nsuch as identifying the exact antibacterial compounds, increasing fermentation\nvolume to obtain higher yield, analyzing LD50 of the extract, adding more MDR\nbacteria tests and performing in vitro toxicity tests. Nevertheless, this study\nadds a valuable insight on the potential of seaweeds-associated bacteria\nespecially from the species of P. aeruginosa<\/em> as the producer of (novel)\nantibacterial compounds. <\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

Authors also thanks The Directorate of Laboratory Management, Research\nFacilities and Science and Technology Park, National Research and Innovation\nAgency (BRIN) Yogyakarta, Indonesia, for SEM and LC-HRMS instrument.<\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

This research was funded by the Fundamental Research Grants awarded by\nthe Ministry of Education, Culture, Research, and Technology Indonesia (fiscal\nyear 2024) to Anak Agung Gede Indraningrat (grant numbers:\n110\/E5\/PG.02.00.PL\/2024, 2927\/LL8\/AL.04\/2024, 565\/Unwar\/DPPM\/PD-13\/2024).<\/p>\n\n\n\n

Conflict of Interest <\/strong><\/p>\n\n\n\n

The author(s) do\nnot have any conflict of interest.<\/p>\n\n\n\n

Data availability<\/strong> Statement<\/strong><\/p>\n\n\n\n

Sequence data of ISP1RL4 bacterial isolate was deposited in GenBank under accession number PP783522. Data related to antibacterial screening, SEM and light microscopy observation, TLC, LC-HRMS and GC-MS can be accessed via the Figshare online repository http:\/\/surl.li\/rpyonh <\/p>\n\n\n\n

Ethics Statement<\/strong><\/p>\n\n\n\n

Experiment described in this manuscript has been ethically approved by\nthe Ethics Commission of Faculty of Medicine and Health Sciences, Warmadewa\nUniversity, Denpasar-Bali under ethics number: 345\/Unwar\/FKIK\/EC-KEPK\/I\/2023 on\n2 October 2023<\/p>\n\n\n\n

Informed Consent Statement<\/strong><\/p>\n\n\n\n

This study did\nnot involve human participants, and therefore, informed consent was not\nrequired<\/p>\n\n\n\n

Clinical Trial Registration<\/strong><\/p>\n\n\n\n

This\nresearch does not involve any clinical trials<\/p>\n\n\n\n

Author Contributions<\/strong><\/p>\n\n\n\n

AAGI designed and provided consumables for experiments, PPCPP performed\nlab works. AAGI wrote draft manuscript. EM performed LC-HRMS and SEM\nexperiments. MDW performed data analysis on GC-MS and LC-HRMS. DAPSM cultured\nMDR bacteria and fermentation of ISP1RL4 isolate. NLPEKS performed TLC and data\nanalysis. All authors read and reviewed the draft manuscript. All authors\nagreed with the manuscript before submission. <\/p>\n\n\n\n

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