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Antibiofilm activity of Morganella morganii JB8F and Pseudomonas fluorescens JB3B compound to control single and multi-species of aquaculture pathogens

BMC Microbiology volume 24, Article number: 381 (2024) Cite this article

Abstract

Background

Indonesia is a country that uses half or more aquatic foods as protein intake. The increased production in aquaculture industries might cause several problems, such as bacterial disease resulting in mass mortality and economic losses. Antibiotics are no longer effective because aquaculture pathogens can form biofilm. Biofilm is a microbial community that aggregates and firmly attaches to living or non-living surfaces. Biofilm formation can be caused by environmental stress, the presence of antibiotics, and limited nutrients. Therefore, it is important to explore antibiofilm to inhibit biofilm formation and/or eradicate mature biofilm. Phyllosphere bacteria can produce bioactive compounds for antimicrobial, antibiofilm, and anti-quorum sensing. Three aquaculture pathogens were used in this study, such as Aeromonas hydrophila, Streptococcus agalactiae, and Vibrio harveyi.

Results

Pseudomonas fluorescens JB3B and Morganella morganii JB8F extracts could disrupt single and multi-species biofilms. Both extracts could inhibit single biofilm formation from one to seven days of incubation time. We confirmed the destruction activity on multi-species biofilm using light microscope and scanning electron microscope. Using GC-MS analysis, indole was the most active fraction of the P. fluorescens JB3B extracts and octacosane from the M. morganii JB8F extract. We also conducted a toxicity test using brine shrimp lethality assay on P. fluorescens JB3B and M. morganii JB8F extracts. P. fluorescens JB3B, M. morganii JB8F, and a mixture of both extracts were confirmed non-toxic according to the LC50 value of the brine shrimp lethality test.

Conclusions

P. fluorescens JB3B and M. morganii JB8F phyllosphere extracts had antibiofilm activity to inhibit single biofilm and disrupt single and multi-species biofilm of aquaculture pathogens. Both extracts could inhibit single species biofilm until seven days of incubation. Bioactive compounds that might contribute to antibiofilm properties were found in both extracts, such as indole and phenol. P. fluorescens JB3B, M. morganii JB8F extracts, and mixture of both extracts were non-toxic against Artemia salina.

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Background

Aquaculture is one of the fastest-growing industries to dominate food production globally. In 2017, global aquaculture production reached 91% (102.9 million tons), mainly in Asia. Until 2017, average fish production increased by 5.7% annually [1]. Indonesia is one country that uses half or more of aquatic foods as protein intake, including fish, crustaceans, molluscs, and aquatic plants. In 2020, Indonesia was ranked second in the world (8.2%) in aquaculture production [2]. The increase in aquaculture industries also faces several problems, such as water pollution, ecosystem damage, and bacterial contamination, that can lead to various bacterial diseases. This can result in economic losses to the aquaculture industry. In 2016, Indonesia recorded the peak of massive fish deaths with financial losses estimated up to IDR 47.25 billion (assuming the price of fish is IDR 10,000 per kg). Mass mortality can reduce aquaculture production by up to 23.5% [3]. Pathogenic bacteria can infect aquaculture products by secreting toxins or by forming biofilms. Treatment of infected aquaculture with antibiotics can cause antibiotic resistance, and antibiotics are no longer effective [4].

Biofilm is a microbial community that interacts and adheres to living or non-living surfaces. Biofilms are formed in response to an unfavourable environment, such as the presence of antibiotics and limiting nutrients as a survival strategy. Microbial interactions can occur between intraspecies and interspecies. Multi-species biofilm can assist microbes in degrading host immune molecules and make biofilm structures more resistant to antibiotics [5]. Biofilms can form in various places, including in aquaculture systems. Pathogenic bacteria infecting fish and other aquaculture products can form biofilms, which is one of the problems in the aquaculture industry nowadays. Aquaculture pathogenic bacteria such as Vibrio harveyi, Streptococcus agalactiae, and Aeromonas hydrophila can form biofilms. Failure in aquaculture may not only be caused by a single pathogen, but probably due by multiple pathogen. Interspecies interaction in biofilm can form multi-species biofilm. Multi-species biofilm with cooperative interactions can form biofilm more fit and more resistant to antibiotics and other disinfectants. Horizontal gene transfer may occur in multi-species biofilm and make bacteria more pathogenic. Coinfection with S. agalactiae and A. hydrophila had been reported in Nile tilapia in Indonesia. The high mortality during the early stages of development often occurs in fish due to a limited immune system and vaccines may no longer be effective in coinfection [6].

Cell communication, namely quorum sensing (QS), can initiate biofilm formation. This mechanism can regulate the expression of several other genes, such as bioluminescence and virulence. This mechanism can be one of the potentials in tackling biofilms on aquaculture pathogens. Bioactive compounds can be used as antibiofilm with the target of inhibiting the mechanism of quorum sensing so that the formation of biofilms is inhibited or destroyed the biofilm. Previous study had been reported that bioactive compounds from Actinomycetes had antibiofilm and anti-quourum sensing agents against single biofilm of aquaculture pathogens. The Actinomycetes extracts had been characterized and the extracts contained the compound which responsible to destruct EPS of biofilm [7]. Phyllosphere bacteria are microbes that can produce many beneficial bioactive compounds including antibiofilm and anti-quorum sensing compounds such as AHL-lactonase in controlling phytopathogens [8]. In a previous study, eight phyllosphere bacteria isolates, including Morganella morganii JB8F and Pseudomonas fluorescens JB3B, can produce antibiofilm and anti-quorum sensing compounds and can destruct biofilm against aquaculture pathogenic bacteria, such as Aeromonas hydrophila, Streptococcus agalactiae, and Vibrio harveyi in a single biofilm. Both extracts can inhibit single biofilm formation in 24 h incubation [9].

This research aimed to identify extracts of P. fluorescens JB3B and M. morganii JB8F that could disrupt multi-species biofilm of aquaculture pathogenic bacteria and identify the duration of biofilm inhibition of P. fluorescens JB3B and M. morganii JB8F extracts against single biofilm of aquaculture pathogenic bacteria used in this study.

Materials and methods

Bacterial cultivation

In this study, phyllosphere bacteria M. morganii JB8F and P. fluorescens JB3B were isolated from previous studies [10]. These bacteria were isolated from Psidium guajava leaves in Karanganyar, Jakarta, Indonesia. Phyllosphere bacteria were inoculated on Brain Heart Infusion Agar (BHIA) (Oxoid) media and incubated at 28 ℃ for 48 h, then refreshed onto King’s B selective media (2 g Protease peptone; 1.5 mL Glycerol; 0.15 g K2HPO4; 0.15 g MgSO4∙7H2O; 20 g Agar; 1 L Distilled water).

In this study, we used Vibrio harveyi and Aeromonas hydrophila from the collection of the Health Aquatic Organism Laboratory of the Department of Aquaculture, Faculty of Fisheries and Marine Science, Bogor Agriculture University. Streptococcus agalactiae was ATCC 27956. A. hydrophila was inoculated on Luria Agar (LA) medium (5 g NaCl; 5 g Yeast extract; 10 g Tryptone; 20 g Agar; 1 L Distilled water) and incubated at 28 ℃ for 24 h. V. harveyi was inoculated on Luria Agar with supplementation of NaCl 2% and incubated at 28 ℃ for 24 h. S. agalactiae was inoculated on Luria Agar and incubated at 37 ℃ for 24 h.

Production of crude extract

Phyllosphere bacteria were extracted by liquid-liquid extraction [11]. The phyllosphere bacteria were cultivated in 100 mL of Luria Broth (LB) (Oxoid) and incubated at 28 ℃ for 48 h at 120 rpm in an orbital shaker. Then, cell suspensions were centrifuged at 7000 x g for 15 min. The supernatant was added to ethyl acetate in equal volume (1:1) and incubated at 28 ℃ for 24 h in an orbital shaker at 120 rpm. Afterward, the solvent layer was evaporated using a rotary evaporator and dried in a vacuum oven at 50 ℃ for 48 h. The extracts were weighed and dissolved with 1% dimethyl sulfoxide (DMSO) to obtain the final 20 mg/mL concentration, then stored at -20 ℃.

Biofilm formation for Destruction and Inhibition Activity

Biofilm formation by aquaculture pathogens used in this study was performed in two experiments based on the activity of antibiofilm, namely destruction and inhibition. The destruction activity was performed on single and multi-species biofilm, while the inhibitory activity was only on single species with different incubation times. Biofilm formation was performed on 96-well microtiter plates. Each aquaculture pathogenic bacteria was cultivated on LB media and incubated at 28 ℃ for A. hydrophila and V. harveyi; and 37 ℃ for S. agalactiae for 24 h, then diluted with sterile LB media to reach 0.132 at OD600.

For inhibition activity, 100 µL of each pathogenic bacteria and 100 µL of phyllosphere extracts (20 mg/mL) were loaded into a 96-well microplate. Then, microplate was incubated for different days (1, 2, 4, or 7 days).

For destruction activity on single-species biofilm, 100 µL of the pathogenic culture was loaded into a 96-well microplate and incubated 28 ℃ for A. hydrophila and V. harveyi; and 37 ℃ for S. agalactiae for 24 h. After incubation, 100 µL of phyllosphere extract (20 mg/mL) was added to each well after planktonic cell were discarded, then re-incubated for 24 h.

For destruction activity on multi-species biofilm, the multi-pathogenic culture was generated by mixing equal volumes of 2 or 3 bacterial isolates (OD600 = 0.132) (Table 1). Thus, four groups were made for multi-species biofilm. A total of 100 µL of the pathogenic culture was loaded into a 96-well microplate and incubated at 28 ℃ for A. hydrophila and V. harveyi; and 37 ℃ for S. agalactiae for 24 h. After incubation, 100 µL of phyllosphere extract (20 mg/mL) was added to each well, then re-incubated for 24 h. A mixture of P. fluorescens JB3B and M. morganii JB8F phyllosphere extracts (20 mg/mL) with the same volume were also tested in multi-species biofilm.

Table 1 Combination of aquaculture pathogen for multi-species biofilm
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Quantification of antibiofilm activity

The effect of antibiofilm from phyllosphere extracts was measured by the crystal violet staining method [12]. The staining process was not performed aseptically. Pathogenic culture without extract was used for positive control, while LB media were used for negative control. After incubation, the planktonic cells and all media were discarded, and the adherent cells were rinsed twice with distilled water, then air-dried for 30 min. A total of 200 µL of 0.4% crystal violet was added to stain the adherent cells and incubated at room temperature for 30 min. After that, the dye was discarded and rinsed five times with distilled water. Then, the 96-well microplates were air-dried for 5 min. A total of 200 µL of ethanol was added to dissolve the stained cells and transferred to a new 96-well microplate. The absorbance of each well was measured with a microplate reader at 595 nm (TECAN M200 PRO). The percentage of inhibition or destruction was calculated using the formula below:

$$\begin{array}{l}\% \;{\rm{Destruction}}\;{\rm{or}}\;{\rm{inhibition}}\;{\rm{activity}}\\\; = \;\frac{{{\rm{OD}}\;{\rm{Positive}}\;{\rm{control}}\;{\rm{ - }}\;{\rm{OD}}\;{\rm{sample}}}}{{{\rm{OD}}\;{\rm{Positive}}\;{\rm{control}}}}\;{\rm{ \times 100\% }}\end{array}$$

GC-MS analysis

The phyllosphere P. fluorescens JB3B and M. morganii JB8F extract were identified by Thermo Scientific™ TRACE™ 1310 gas chromatography systems using TG-5MS GC column (30 m length, 0.25 mm diameter, 0.25 μm width). The extracts were dissolved in ethyl acetate in a 1:1 (w/v) ratio and injected 1 µL. The carrier gas was helium at a 24 mL/min flow rate. The initial column temperature was 60 ℃ and gradually increased to 325 ℃ at 10 ℃/min. The detected compounds were reflected in peaks on the chromatogram [13] .

Toxicity assay using brine shrimp lethality assay (BSLA)

The toxicity of phyllosphere extracts, P. fluorescens JB3B, M. morganii JB8F and a mixture of both was tested using Brine Shrimp Lethality Assay (BSLA) method with some modifications [14]. A total of 3 mg of brine shrimp (Artemia salina) eggs were hatched in 500 mL of artificial seawater (38 g NaCl, 1 L distilled water) under constant aeration and illumination for 24 h. The extracts were dissolved in artificial seawater for a stock solution. Ten nauplii were collected using a pipette and transferred to test tubes filled with 4.5 mL of artificial seawater. Then, 500 µL of extracts was added to each tube. The extracts were diluted from stock solution to various final concentrations (10, 100, 500, and 1000 ppm). A total of 500 µL of K2Cr2O7 was used for positive control, while negative control was artificial seawater. The tubes were incubated at room temperature for 24 h, and the dead nauplii were counted. The death percentage of nauplii was calculated using the formula below:

$$\:\text{\%\:Death=}\:\frac{\text{The\:number\:of\:dead\:nauplii}}{\text{Total\:nauplii}}\text{\:x\:100\%}$$

Then, LC50 was calculated using simple linear reggresion model.

Scanning Electron microscopy (SEM) and light microscopy analysis

Scanning Electron Microscope (SEM) was performed at BRIN, Indonesia. Microscopy analysis was determined destruction activity of P. fluorescens JB3B extract as the highest antibiofilm activity against multi-species biofilm in more detail. Each aquaculture pathogen was grown in broth media at 28 ℃ for A. hydrophila and V. harveyi; and 37 ℃ for S. agalactiae for 24 h, then diluted to reach 0.132 at OD600. The multi-pathogenic culture was generated by mixing equal volumes of 2 or 3 bacterial isolates (OD600 = 0.132). One mL of multi-pathogenic culture was grown in 1 × 1 cm cover glass at 28 ℃ for A. hydrophila and V. harveyi; and 37 ℃ for S. agalactiae for 24 h. After incubation, the cover glass was transferred into a new sterile petri dish with 500 µL extracts, then re-incubated for 24 h. A total of 500 µL of multi-pathogenic culture was used for positive control. For preparation of SEM, planktonic cell was discarded and the biofilm was added with 2.5% (v/v) of glutaraldehyde dissolved in distilled water, and the fixed biofilm was incubated at 4 ℃ for 24 h. The fixed biofilm was dehydrated with 30%, 50%, 70%, 96%, and 100% (v/v) alcohol for 15 min each concentration and after dehydrated all concentration, fixed biofilm was dried at 28 ℃ for 10 min. For light microscopy, the cover glass was rinsed twice with distilled water and added with 0.4% (w/v) of crystal violet which dissolved in ethanol absolute for 10 min. Then, the cover glass was rinsed with distilled water and observed under light microscopy [13] .

Statistical analysis

The data was analyzed using ANOVA. All statistical analysis were performed using Statistical Package for the Social Science (SPSS) software version 25. All graphs were constructed using Microsoft Excel (Microsoft 365).

Results

Quantification of biofilm destruction activity

Both extracts showed biofilm destruction activity against a single pathogen used in this study. Extracts of P. fluorescens JB3B and M. morganii JB8F had similar activity in destroying A. hyrdophila and V. harveyi biofilm. The destruction activities were 59.70% by P. fluorescens JB3B extract and 53.76% by M. morganii JB8F extract against A. hydrophila and 27.49% by P. fluorescens JB3B extract and 25.33% by M. morganii JB8F against V. harveyi. Otherwise, the extract of P. fluorescens JB3B had a higher destruction activity than M. morganii JB8F extract against biofilm of S. agalactiae. The destruction activity against S. agalactiae biofilm for P. fluorescens JB3B extract was 31.17% and 20.23% for M. morganii JB8F extract (Fig. 1a). Extracts of P. fluorescens JB3B and M. morganii JB8F significantly destruct single biofilm pathogen aquaculture (p < 0.05).

In the multi-species biofilm, the extract of P. fluorescens JB3B had higher destruction activity than M. morganii JB8F. The mixture of P. fluorescens JB3B and M. morganii JB8F extracts could disrupt multi-species biofilms and had higher activity than M. morganii JB8F extract. The highest destruction was 56.41% for P. fluorescens JB3B against three mixtures of aquaculture pathogen biofilms. The lowest destruction activity was 18.99% for M. morganii JB8F against V. harveyi and S. agalactiae biofilms (Fig. 1b; Table 2). Extracts of P. fluorescens JB3B and M. morganii JB8F significantly destruct multi-species biofilm pathogen aquaculture (p < 0.05).

Fig. 1
figure 1

Percentage of biofilm destruction activity against (a) single aquaculture pathogen, A. hydrophila, V. harveyi, and S. agalactiae (p < 0.05), and (b) multi-species pathogen (P < 0.05). The data were analyzed by one way ANOVA. (Vertical bars are standard errors, *: significantly different at p < 0.05)

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Table 2 Biofilm destruction activity (%) against single and multi-species aquaculture pathogens
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Quantification of biofilm inhibition activity

Both P. fluorescens JB3B and M. morganii JB8F extracts had ability to inhibit the formation of biofilms for the duration of 1 to 7 days of incubation time. The extracts had the lowest activity in 2 days of incubation time against biofilm of all aquaculture pathogens, except A. hydrophila in 4 days of incubation. Then, the inhibition activity increased and performed the highest inhibition activity in 7 days of incubation time against biofilm of all pathogens, except V. harveyi. Extract of P. fluorescens JB3B had higher inhibition activity than M. morganii JB8F extract (Fig. 2). The lowest inhibition was 42.41% by M. morganii JB8F in 4 days and the highest inhibition was 86.38% by P. fluorescens JB3B in 7 days against A. hydrophila biofilm (Table 3). The lowest inhibition was 47.23% by M. morganii JB8F in 2 days and the highest inhibition was 75.16% by JB3B in 24 h against V. harveyi biofilm (Table 4). The lowest inhibition was 40.01% by M. morganii JB8F in 2 days incubation and the highest inhibition was 87.27% by P. fluorescens JB3B in 7 days incubation against S. agalactiae biofilm (Table 5). Both P. fluorescens JB3B and M. morganii JB8F extracts significantly inhibit single biofilm against A. hydrophila, V. harveyi, and S. agalactiae in 24 h, 2, 4, and 7 days (p < 0.05). However, the extracts didn’t significantly differ in biofilm inhibition activity against single biofilm pathogen aquaculture in various incubation time (p > 0.05).

Fig. 2
figure 2

Percentage of biofilm inhibition activity against single aquaculture pathogen biofilm (a) A. hydrophila, (b) V. harveyi, and (c) S. agalactiae with different incubation times. The data were analyzed by one way ANOVA. (Vertical bars are standard errors, *: significantly different at p < 0.05)

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Table 3 Inhibition activity (%) against A. hydrophila biofilms
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Table 4 Inhibition activity (%) against V. harveyi biofilms
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Table 5 Inhibition activity (%) against S. agalactiae biofilms
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GC-MS analysis

GC-MS analysis showed that extracts of P. fluorescens JB3B and M. morganii JB8F contained specific bioactive compounds. The major compounds of P. fluorescens JB3B performed with some of highest peak including: (1) Phenol, with a retention time of 4.28; (2) Phenylethyl alcohol, with aretention time of 6.17; (3) Indole, with a retention time of 8.71. The major compounds present in M. morganii JB8F extract were: (1) Phenylethyl alcohol, with a retention time of 6.17; (2) Pyrrolo[1,2-a]pyrazine-1,4 -dione, hexahydro-3-(2-methylprop yl)-, with a retention time of 16.04; (3) Octacosane, with a retention time of 20.20 (Fig. 3, Supplementary Tables 1 and Supplementary Table 2).

Fig. 3
figure 3

GC-MS chromatogram of the bioactive compounds from (a) P. fluorescens JB3B extract and (b) M. morganii JB8F extract

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Toxicity assay using brine shrimp lethality assay (BSLA)

Extracts of P. fluorescens JB3B, M. morganii JB8F, and mixture of both extracts were tested using BSLA method to calculate LC50 value. This assay was performed in triplicates with four different concentrations of extracts. All the extracts had LC50 value more than 1000 µg/mL, which means these extracts were considered as non-toxic (Table 6).

Table 6 LC50 value with BSLA method
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Scanning Electrone Microscopy (SEM) and light microscopy analysis

The destruction of biofilm with P. fluorescens JB3B extract were observed using SEM (Fig. 4) and light microscopy (Supplementary Fig. 1). SEM showed biofilm formation more compact on all positive controls compared to biofilm formation with P. fluorescens JB3B extract treatment.

Fig. 4
figure 4

Biofilm destruction activity on SEM VH + AH (a) positive control, (b) treated with P. fluorescens JB3B extract; VH + SA (c) positive control, (d) treated with P. fluorescens JB3B extract; AH + SA (e) positive control, (f) treated with P. fluorescens JB3B extract; VH + AH + SA (g) positive control, (h) treated with P. fluorescens JB3B extract

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Discussion

The phyllosphere is a community of bacteria living on plant surfaces, especially leaf surfaces. The host plant can shape the composition of the phyllosphere community [15]. Phyllosphere bacteria can also produce secondary metabolites, such as antimicrobial and antibiofilm compounds. Antibiofilm compounds produced by the phyllosphere can inhibit the quorum sensing mechanism by degrading the AHL signal molecule with the lactonase enzyme [16]. P. fluorescens JB3B and M. morganii JB8F phyllosphere bacteria were isolated from Psidium guajava leaves which were known to possess antimicrobial and antibiofilm properties [16]. According to 16 S rRNA gene from previous study, isolate P. fluorescens JB3B had 92% similarity to Pseudomonas fluorescens UTB 111 with GenBank accession number OM763955. Isolate M. morganii JB8F had 97% similarity to Morganella morganii NBRC 3848, and the GenBank accession number OL960630. P. fluorescens UTB 111 had hydrolytic activity, such as protease and amylase [17].

Phyllosphere extracts of P. fluorescens JB3B and M. morganii JB8F had antibiofilm activities, either inhibiting or disrupting all tested single aquaculture pathogens, such as A. hydrophila, V. harveyi, and S. agalactiae. These results were comparable to previous studies. Biofilm inhibition may occur because both P. fluorescens JB3B and M. morganii JB8F extracts produced anti-quorum sensing compounds [9, 18]. Anti-quorum sensing mechanisms can be achieved in various alternative ways, such as inactivation of signal molecules, blocking receptor signals using analog compounds to signal molecules, and blocking the formation of receptor signals [19]. Biofilm formation begins with an initial attachment to the surface, forming microcolonies and extracellular polymeric substances (EPS), maturation, and dispersion. EPS contains polysaccharides, nucleic acid, and protein. The composition of EPS in each bacterium is different. Disruption of biofilm may occur because the extracts could destroy the composition of EPS in the biofilm [20]. Biofilm formation is influenced by microbial interactions and bacteria communication, called quorum sensing. Quorum sensing is a way for bacteria to communicate between cells. Quorum sensing is the ability to produce and respond to extracellular signal molecules at a spesific cell density [21] .

Biofilm formation and development of A. hydrophila is mainly regulated by ahyI/R quorum sensing systems with C4-AHL and C6-AHL signal molecules. The previous study showed that disrupting ahyI/R systems could decrease the motility of A. hydrophila. This pathogen has two types of flagella, polar and lateral. Motility is essential to help bacteria colonize and make biofilms [22]. Besides biofilm formation, A. hydrophila can affect haemorrhagic septicemia and virulence factor (enterotoxins and haemolysis) in fish, such as common carp and tilapia, through QS systems. A previous study showed that biofilm formation in common carp (Cyprinus carpio) caused phagocytic, neutrophil, and bactericidal activity of fish blood cells to increase. But, this immune response could not defeat the infection, which results in mass mortality in fish [23].

Biofilm formation of V. harveyi is regulated by quorum sensing through the luxR QS system. The signal molecules used in biofilm formation by V. harveyi are Harveyi Autoinducer 1 (HAI-1), Autoinducer 2 (AI-2), and Cholera Autoinducer 1 (CAI-1). These signal molecules have different functions. HAI-1 essentially uses intraspecies communication, CAI-1 is used for inter-Vibrio communication, and AI-2 is produced for interspecies communication [24]. Biofilm can be found in the gastrointestinal tract and hepatopancreas of infected shrimp [25]. Chitin is the key for the attachment stage to form biofilm for adhesion and colonization of these bacteria. Besides that, nitric oxide (NO) was reported to contribute to light production through the NOX/HqsK quorum sensing. NO regulates flagella production and enhances biofilm production through LuxU/LuxO/LuxR pathway [26]. Besides infecting shrimps, V. harveyi can infect mollusks and fishes to cause vibriosis. Fish infection usually occurs from contaminated water and contact with the digestive tract or skin. Besides that, V. harveyi can form biofilm on the walls of rearing tanks, enhancing the risk of vibriosis outbreaks [27].

S. agalactiae can form biofilms in the brain associated with meningoencephalitis in fish. Quorum sensing is a way for bacteria to regulate sporulation, induction of virulence factor, and biofilm [28]. Biofilm formation in S. agalactiae is controlled by the regulation of the agr system. The quorum sensing system consists of three components: signal peptide, histidine kinase sensor, and response regulator. Phosphorylated agrA activates the regulatory protein SarA that controls biofilm formation. Besides that, QS in these bacteria is also used for virulence factors, such as the expression of 2 polysaccharide antigens, group B with rhamnose units, and capsular polysaccharide. The production of rhamnose is critical in the biofilm development of S. agalactiae [29]. Motility is essential for biofilm formation. These bacteria form pili, encoded by GBS pilus machinery. The genes encoding the GBS pilus are clustered in three genomic islands (Islands PI-1, -2a, and − 2b) [30].

P. fluorescens JB3B and M. morganii JB8F extracts performed destruction activity against multi-species biofilm, with the highest destruction activity for P. fluorescens JB3B with the percentage of 56.41% against three mixtures of aquaculture pathogens biofilm. All extract samples have the lowest destruction activity against multi-species biofilm of two species which were V. harveyi and S. agalactiae. Destruction activity for M. morganii JB8F was 18.99% against multi-species biofilm of V. harveyi and S. agalactiae (Table 2). The mechanism of destruction biofilm can occur enzymatic by destroying EPS of biofilm. P. fluorescens JB3B extract had the highest destruction activity against multi-species biofilm. A mixture of both extracts had lower destruction activity than P. fluorescens JB3B extract; both extracts might contain compounds that can inhibit each extract, so the destruction activities were lower than only P. fluorescens JB3B.

Interspecies interactions can form multi-species biofilms in which bacterial adhesion proteins play an essential role in coaggregation during biofilm formation. Interactions in multi-species biofilms can occur competitively by forming other microbial inhibitor compounds, but there are also cooperative interactions, such as to maintain biofilm fitness. For example, type IV pili and flagella of P. aeruginosa are used to compete with immature Agrobacterium tumefaciens and have the advantage of growing in multi-species biofilm [31].

In a previous study, A. hydrophila biofilm formation was reduced by P. aeruginosa and P. fluorescens. Both bacteria P. aeruginosa and P. fluorescens could attach to the surface, forming very compact and strong biofilms. Besides that, P. aeruginosa and P. fluorescens produced exoprotease that can destroy the EPS of A. hydrophila biofilms [32]. S. agalactiae could form multi-species biofilms with S. mutans synergistically. GtfB and GtfC of S. mutans are responsible for EPS production, which plays essential roles in interspecies coaggregation [33]. V. harveyi had competitive interactions with V. fischeri. Bacteria V. fischeri was known produces aerobactin to compete under iron-limiting conditions [34].

Literature of multi-species biofilms between aquaculture pathogens tested in this study are still limited. According to positive control, all multi-species biofilm tested could form biofilm. Interactions between bacteria need to be studied further. Co-infection between aquaculture pathogens tested had been reported. Co-infection of A. hydrophila and S. agalactiae occurred in tilapia, resulting in 70% mass mortality. Mainly, co-infection between these bacteria infects the liver. A. hydrophila and S. agalactiae might have synergistic effects resulting in increased severity of the disease [35].

P. fluorescens JB3B and M. morganii JB8F extracts had inhibition activity until seven days of incubation time. The highest inhibition activity performed on seven days against biofilm of A. hydrophila and S. agalactiae. According to a previous study, A. hydrophila formed biofilm maximum at 24 h until 48 h and decreased at 72 h. The crude extracts had similar inhibition activity from one day of incubation time until two days of incubation against A. hydrophila biofilms [32]. Biofilm formation of V. harveyi was stabilized until four days in the presence of chitin. Chitin was used for adhesion. Biofilm inhibition activity was found to decreased at two days and increased at 4 days [36]. In the previous study, a biofilm of S. agalactiae was optimum after 48 h of incubation. This was appropriate with the results where the inhibition activity showed similar at one day and two days of incubation time [37]. P. fluorescens JB3B and M. morganii JB8F extracts had anti-quorum sensing activity and can inhibit pigment production in Chromobacterium violaceum. Anti-quorum sensing can inhibit the bacteria from attaching to the surface so that biofilm formation is disrupted [8].

GC-MS analysis showed that extract of P. fluorescens JB3B had major indole compounds. Indole is an alkaloid with many biological activities, such as anti-microbial, anti-inflammatory, antioxidant, and plant growth regulators [38]. Indole is produced by the degradation of tryptophan by tryptophanase. Indole is abundantly available in natural resources and produced by many bacteria. Escherichia coli can produce indole and acetate during the stationary phase. This molecule had been reported to have antibiofilm activity. Indole can inhibit motility and biofilm formation by disturbing AHL signal molecules and the QS regulator of P. putida [39]. In the other study, indole and its derivatives, such as 3-indole acetonitrile, 5-fluoro indole, and 6-fluoro indole, had the potential to inhibit the biofilm formation of S. marcescens by interfering with QS and motility. The other indole derivatives, 6-fluoro indole, and 7-methyl indole, could interfere with yeast’s extracellular polymeric substance production (EPS) [40].

The other bioactive compound found in P. fluorescens JB3B and M. morganii JB8F extracts is phenyl ethyl alcohol. Phenylethyl alcohol is an aromatic alcohol and volatile organic compound. Unfortunately, the literature on phenyl ethyl alcohol-producing bacteria is still limited compared to plant extract. Antibiofilm and antibacterial activities were found in burdock leaf extracts’ essential oil. The high antibiofilm and antibacterial activities are the combined action of phenyl ethyl alcohol, benzyl alcohol, borneol, and the other compounds in the essential oil. Biofilm formation of S. aureus and E. coli were fully inhibited with the concentration of 250 and 500 µg/mL [41]. In a previous study, phenyl ethyl alcohol was detected in Diaporthe phaseolorum SSP12 plant extracts. This plant extract showed anti-quorum sensing, antibiofilm, and antioxidant activities against P. aeruginosa PAO1 [42].

Phenol was also found in P. fluorescens JB3B extract. Plants produce many phenolic compounds, such as tannins, flavonoids, and anthocyanins. Eugenol and cinnamaldehyde have been reported to inhibit the biofilm formation of L. monocytogenes. Flavonoid is a group of phenolic products that have antibacterial and antibiofilm activity. Naringenin and quercetin are flavonoids that have anti-quorum sensing activity against V. harveyi and inhibit biofilm formation of V. harveyi and E. coli [43]. Essential oil florfenicol, which contains a phenol compound, has been shown to have antibacterial and antibiofilm properties against A. hydrophila [44]. Plant extracts from tropical fruit, Syzygium cumini that contain phenolic had been reported to inhibit quorum sensing and have antibiofilm and antimicrobial properties against pathogen, such as E. coli, P. fluorescens, and L. monocytogenes. These extracts inhibited swarming motility and inhibited biofilm formation of A. hydrophila [45]. Tannic acid was also reported to have anti-quorum sensing properties against A. hydrophila by inactivating signal molecules [46]. Unfortunately, the literature on phenol against S. agalactiae biofilm is still limited. But, phenolic compounds from extracts of edible fungi have been reported to have antimicrobial and antioxidant properties against S. aureus, S. agalactiae, and Candida albicans [47].

Pyrrolo[1,2-a]pyrazine-1,4 -dione, hexahydro-3-(2-methylprop yl)- was detected in the Morganella morganii JB8F extracts. This compound is known as Cyclo(Leucyl-Prolyl) and 3-Isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione. In the previous study, Staphylococcus xylosus VITURAJ10 extracts produced this compound and have anticancer and antibacterial properties against E. coli, S. enterica, and S. aureus [48]. Streptomyces sp. UPMRS4 also produced this compound and had antifungal activity against Pyricularia oryzae, causing the agent of rice blast disease [45]. This compound had been reported to have antibiofilm properties against biofilm forming Gram negative bacteria. Endophytic actinomycetes Nocardiopsis sp. GRG 1 (KT235640) extract produced this compound and inhibited the biofilm formation of P. mirabilis and E. coli [49]. The other endophytic bacteria from red sea soft corals could produce secondary metabolites with antibiofilm properties. The compounds detected, such as pyrrolo[1,2-a]pyrazine-1,4 -dione, hexahydro-3-(2-methylprop yl)- and heneicosane could inhibit biofilm formation that causes biofouling [50]. Heneicosane was also detected on M. morganii JB8F extracts.

Octacosane is long chain alkane hydrocarbons. This compound was found only on M. morganii JB8F extracts. Octacosane was extracted from medicinal herb leaves and had anti-cancer properties [51]. In the previous study, octacosane was found in the Mantidis ootheca extracts with antimicrobial and antibiofilm properties against P. aeruginosa. Besides octacosane, M. ootheca extracts were also detected as heptacosane and hentriacontane, which have antimicrobial and antibiofilm properties [52]. Heptacosane was detected on P. fluorescens JB3B extracts with a retention time of 20.96, and hentriacontane was detected on M. morganii JB8F extracts with a retention time of 21.76. Octacosane was also detected in plant extracts from Trapa natans L. (12.4%). It had been reported to have antimicrobial and antibiofilm properties with other compounds, such as phenolic, flavonoid, and heptacosane against P. aeruginosa and E. coli [53].

Brine shrimp lethality assay is simple, rapid, and inexpensive. This assay determines toxicity based on the lethality of brine shrimp (Artemia salina) with exposure to crude extract with various concentrations. The toxicity of crude extract is established according to the LC50 value. The extract is considered non-toxic if the LC50 value is more than 1000 µg/mL. The extract is considered weak toxic if the LC50 value is between 500 and 1000 µg/mL. If the LC50 value is less than 1000 µg/mL, the extract is considered toxic [54]. In this study, all of the extract samples were consider as non-toxic against A. salina because the LC50 value is more than 1000 µg/mL (Table 6).

The destruction activity against multi-species biofilm was confirmed using light microscopy and SEM. Multi-species pathogens could form biofilms synergistically on cover glass according to positive control. The biofilms treated with P. fluorescens JB3B extract were significantly different from the control (Fig. 4, Supplementary Fig. 1).

Limitation

This study only performed a preliminary toxicity test; further study is needed to complete a more advanced toxicity assay applicable to aquaculture products. In addition, this study only quantified antibiofilm for several aquaculture pathogens, further study need to be conducted on antibiofilm activities against other aquaculture pathogens that can form multi-species biofilm in aquaculture products, such as fish and shrimps.

Conclusion

This study found that P. fluorescens JB3B and M. morganii JB8F extracts had antibiofilm properties to disrupt biofilm against multi-species tested aquaculture pathogens. Both extracts could inhibit single species biofilm from 24 h to 7 days of incubation time. We also analysed P. fluorescens JB3B and M. morganii JB8F compounds using GC-MS. Indole, phenol, pyrrolo[1,2-a]pyrazine-1,4 -dione, hexahydro-3-(2-methylprop yl)-, and other compounds might contribute to antibiofilm activities against aquaculture pathogens. The phyllosphere extracts of P. fluorescens JB3B and M. morganii JB8F could be potential candidates for antibiofilm agents in the aquaculture industry. Further research is needed to identify mechanism of disruption and against other aquaculture pathogens in multi-species biofilms and toxicity assay more advance.

Data availability

All data deposited in the Genebank is publicly available with Genbank accession number OM763955.1 (https://www.ncbi.nlm.nih.gov/nuccore/OM763955.1/) and OL960630.1 (https://www.ncbi.nlm.nih.gov/nuccore/OL960630). All data generated or analized during this study are included in this published article and an additional file (Additional File 1).

Abbreviations

AHL:

Acyl-homoserine lactones

GC-MS:

Gas chromatography-mass spectrometry

GBS:

Group B Streptococcus

LC50 :

Lethal concentration 50

SEM:

Scanning electron microscopy

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Funding

The study received funding from Kemdikbud 2023. However, the funder had no involvement in the study’s design, data collection, analysis, interpretation, and writing of the manuscript.

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Authors and Affiliations

  1. Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, Jalan Raya Cisauk- Lapan No. 10, Sampora, Cisauk, Tangerang, Banten, 15345, Indonesia

    Valencia Vanessa, Diana Elizabeth Waturangi & Adi Yulandi

  2. Department of Aquatic Resources Management, Faculty of Marine Science and Fisheries, University of Udayana, Denpasar, Bali, 80361, Indonesia

    Pande Gde Sasmita Julyantoro

  3. Department of Biology Education, Faculty of Teacher Training and Education, Khairun University, Ternate, 97728, Indonesia

    Nurmaya Papuangan

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Contributions

V.V. Conducted the research and data analysis, prepared proposal writing and manuscript under advisory of D.E.W. and A.Y. A.Y. and P.G.S.J. and N.P. Data analysis and manuscript preparation. D.E.W. Data analysis, Research design and person in charge of all the research project. All authors read and approved the final manuscript.

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Vanessa, V., Waturangi, D.E., Yulandi, A. et al. Antibiofilm activity of Morganella morganii JB8F and Pseudomonas fluorescens JB3B compound to control single and multi-species of aquaculture pathogens. BMC Microbiol 24, 381 (2024). https://doi.org/10.1186/s12866-024-03544-6

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