Freshly caught squids were bought from a local wet market in Al Ain, United Arab Emirates. The squids were placed in an ice box and transported immediately to the laboratory. The squids were dissected, and the ink sac was identified. The ink sac was gently opened using a sterile scalpel and the ink was collected by absorption using a sterile cotton swab and aseptically transferred into a test tube containing 0.85% saline solution. The solution was then serially diluted up to 10^–4 and 0.1 mL was spread plated onto sea water agar (SWA; HiMedia^®) plates and incubated at 28°C for 48 h. The spread plating was conducted in duplicates. After incubation, each plate was visually observed in the dark for LB colonies. The plates without LB colonies were further incubated for another 24 h. To obtain pure isolates, an LB colony from each plate was picked using an inoculating needle, streaked onto a new SWA plate, and observed after incubation at 28°C for 48 h. The pure LB isolates were then subjected to bioluminescence screening.

To determine the bioluminescence intensity and select the most suitable isolates for further study, 14 LB isolates were subjected to bioluminescence screening (Bao et al., 2023). The positive control was Escherichia coli DH108 transformed with plasmid pJE202 expressing the Lux operon genes from Vibrio fisheri. Overnight cultures of the LB isolates were incubated on SWA plates at 28°C for 48 h. E. coli DH108 was incubated on a plate containing Luria–Bertani agar with ampicillin at 35°C for 48 h. After incubation, colonies were picked from the plates and transferred to a test tube containing 0.85% saline solution. The cell density of each suspension was determined with reference to the 0.5 McFarland standard (∼1.5 × 10⁸ CFU/mL). A loopful (HiMedia^® Hi-FlexiLoop 2; 2.0 mm in diameter, calibrated to 0.005 mL) of each suspension was inoculated in tubes containing 10 mL artificial sea water broth (HiMedia^®) with 0.25% [w/v] yeast extract (HiMedia^®) and 0.5% [w/v] tryptone type-1 (HiMedia^®) (ASW-YE-T) and incubated at 28°C while shaking (120 rpm) in a shaking incubator (Bioevopeak Co., Ltd., shaking incubator) for 48 h. For E. coli DH108, Luria–Bertani broth with ampicillin was used. After incubation, 200 µL of inoculum from each culture was transferred to a well of a microtiter plate (black, flat bottom, 96 wells, Costar^®), with nine replicates. The microtiter plate was covered with foil and left to stand for 10 min before reading. The bioluminescence intensity was measured using a GloMax^® Discover Microplate Reader (Promega Corporation, Madison, WI, USA) with a 10-s integration period prior to reading. Bioluminescence is reported as specific bioluminescence (SB), which is the log-transformed values relative light unit (RLU). As a result, two LB strains were selected: LB01 and LB09. Stock cultures of these bacteria were maintained in cryovials containing 20% [w/v] NaCl–trypticase soy broth with 20% [v/v] glycerol and stored at − 20°C for further study.

Microbeads, which made of polyethylene with inorganic metal pigments, extracted from cosmetic products by Habib et al. (2020) were used in this study. First, 10 g of microbeads were rinsed using type 1 ultrapure water to remove unwanted particulates, submerged in 70% ethanol for 30 min, washed with deionized water, and dried at 45°C in a hot air oven (Daihan Scientific Co., South Korea). The beads were ground using a sterile pestle and mortar and then exposed to UV light overnight. A small amount (0.01 g) of the GMPs were observed under a stereomicroscope (Leica Zoom 2000). The size of GMPs was measured using the ImageJ software (https://imagej.net/ij/) and the size distribution was determined (Supplementary Figure S1). To ensure asepsis, 0.1 g of GMPs was transferred into several tubes containing trypticase soy broth and incubated at 35°C for 48 h. After incubation, no tube showed turbidity.

The Phb. leiognathi strains in stock cultures were revived in tubes containing ASW-YE-T broth and incubated at 28°C for 48 h. Revived bacterial cells were collected by repeated centrifugation (6,000 × g for 10 min) and resuspended in phosphate-buffered solution (PBS, pH 7.4). The optical density at 600 nm (OD₆₀₀) was determined and adjusted to 0.5–0.8 (Swift et al., 1993; Ahmad et al., 2012; Muneeswaran et al., 2021). To initially determine the optimal temperature for peak bioluminescence, an experiment was conducted across a temperature range of 15°C–35°C. After 48 h of incubation, bioluminescence intensity was measured at each temperature, revealing that the highest levels were observed at 22°C and 30°C. The said temperatures were selected for this study to characterize growth kinetics and bioluminescence of Phb. leiognathi strains.

Freshly revived bacterial cells were collected, and new suspensions were prepared. Subsequently, 3 µL of the suspension was inoculated into tubes containing 15 mL ASW-YE-T broth and incubated at 22°C or 30°C while shaking (120 rpm). The bacterial growth was monitored over time (until it reached the death phase) by transferring 200 µL to a microtiter plate well and measuring the OD₆₀₀ using a GloMax^® Discover Microplate Reader (Promega Corporation, Madison, WI, USA).

To investigate the effects of GMPs on Phb. leiognathi strains, fresh cultures of the strains in the exponential growth phase were prepared. An aliquot was transferred into a microcentrifuge tube and the cells were harvested by centrifugation (6,000 × g for 10 min). The supernatant was carefully discarded, and the pellet was washed twice with PBS (pH 7.4) and then centrifuged again (6,000 × g for 10 min). After washing twice with PBS (pH 7.4), the supernatant was carefully discarded, ensuring the pellet remained undisturbed. The OD₆₀₀ was adjusted to 0.5–0.8 and the bacterial suspension was immediately used for the GMP exposure experiment. Various GMP concentrations (0.25%, 0.50%, 1.00%, and 2.00% [w/v] per mL) were added to tubes containing 15 mL of ASW-YE-T broth. Control tubes contained ASW-YE-T broth without GMPs. Next, 10 µL of the bacterial suspension was transferred into each tube. The tubes were then incubated at 22°C or 30°C while shaking (120 rpm) in a shaking incubator (Bioevopeak Co., Ltd.). The bioluminescence responses of the bacteria to different GMP concentrations and temperatures were monitored over two timeframes: (1) every 5 h for 3.1 days (75 h) to assess immediate effects, and (2) every 24 h for 7 days to assess long-term effects. To prevent nutrient depletion, double-strength ASW-YE-T broth was added in the 7-day experiment. To assess the bioluminescence, 200 µL (n = 3) from each tube was transferred to each well of a black microtiter plate. The plate was covered with foil and left undisturbed for 10 min. SB was then measured as described in the Bioluminescence Screening section. This experiment was conducted three times on separate occasions, with triplicate wells (n = 9).

This experiment was conducted to determine whether Phb. leiognathi strains previously exposed to GMPs for 3.1 days and 7 days can recuperate based on their bioluminescence intensities. A 1-mL aliquot of the bacterial culture was carefully pipetted from the tubes in 3.1-day and 7-day GMP exposure experiment, transferred into a microcentrifuge tube, and centrifuged (6,000 × g for 10 min). Next, the supernatant was carefully discarded, the pellet was washed three times with PBS (pH 7.4), and then resuspended in PBS (pH 7.4). The OD₆₀₀ was measured, adjusted to 0.5–0.8, and 10 µL of each prepared bacterial suspension was transferred into tubes containing 15 mL ASW-YE-T broth. The tubes were then incubated for 35 h at 22°C or 30°C while shaking (120 rpm). The bioluminescence responses of the strains were monitored every 5 h for 35 h. Then, 200 µL (n = 3) from each tube was transferred to the wells of a black microtiter plate. The plate was covered with foil and left undisturbed for 10 min. Next, the SB was measured as described in the Bioluminescence Screening section. This experiment was conducted three times on separate occasions, with triplicate wells (n = 9).

The cell viability of Phb. leiognathi strains after exposure to varying GMP concentrations were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays with modifications (Wang et al., 2010). To prepare the MTT solution, 50 mg of MTT powder (bioWORLD, GeneLinx International, Inc. USA) was dissolved in 10 mL deionized water while stirring at 37°C. The solution was stored in microcentrifuge tubes at − 20°C until use. 3 μL of each bacterial suspension was inoculated in a well of a microtiter plate (clear, flat bottom, 96 wells, Costar^®) containing 100 µL of ASW broth. The plate was incubated at 22°C or 30°C for 8 h. Next, 10 µL of MTT solution was added using a multi-channel pipette (Eppendorf Research^®) and left to stand for 20 min. Thereafter, the broth was removed by gentle pipetting and 100 µL of dimethyl sulfoxide (purity >99%; Merck, Darmstadt, Germany) was added. The OD₅₆₀ was measured using a microtiter plate reader after shaking the plate for 10 s. Each OD value was corrected based on the blank (medium without cells), and the mean corrected OD values were then calculated. This experiment was conducted with replicates (n = 12).

The biofilm formation of Phb. leiognathi strains after GMP was assessed using the method (De Jesus and Dedeles, 2020) with modifications. To initiate biofilm formation, 1% (w/v) squid’s ink solution was used to coat the wells of a microtiter plate (clear, flat bottom, 96 wells, Costar^®). The ink was collected from a squid’s ink sac using a sterile syringe and added to sterile deionized water. The ink solution was heated at 55°C for 10 min and then 100 µL was transferred to the wells and the microtiter plate was placed in an incubator overnight. Next, the contents were removed from the wells and 100 µL of double-strength ASW-YE-T broth was added to the wells. The wells were then inoculated with 3 µL of bacterial suspension and incubated at 22°C or 30°C while shaking (150 rpm) for 48 h. After incubation, the broth was removed by gentle pipetting, replenished with freshly prepared ASW-YE-T to support the biofilm formation, and incubated again at the previously mentioned temperatures for 48 h. The growth in microtiter plate wells was assessed based on the OD₆₀₀ using the GloMax^® Discover Microplate Reader. Using a multi-channel pipette (Eppendorf Research^®), the broth of each well was removed, and the wells were gently washed once with 200 µL of sterile PBS (pH 7.2), and then air-dried for 20 min. The attached biofilms in the wells were stained with 130 µL of 1% (v/v) crystal violet solution (HiMedia^®) for 5 min and washed three times with 200 µL of sterile distilled water. The stained biofilms in the wells were solubilized with 130 µL of absolute ethanol (Carlo Erba, Milan, Italy) and the OD₅₆₀ was assessed. The results were expressed as specific biofilm formation (SBF), calculated as (A – B)/C, where A is the OD₅₆₀ of the stained biofilms, B is the OD₅₆₀ of the stained blank control wells (to eliminate non-specific or abiotic OD values), and C is the OD₆₀₀ of bacterial growth in ASW-YE-T broth. The assay was performed in replicates (n = 9).

The bioluminescence intensities were normalized (min-max scaling; 0–1) to reduce potential errors. The differences in mean OD values in MTT assays and the mean SBF in biofilm formation assays (control groups vs. exposed groups) were examined using two-way ANOVA, followed by Dunnett’s post hoc analysis in Graphpad Prism 10 (version 10.2.3). p < 0.05 was accepted as statistically significant, indicating reliable differences between control groups and exposed groups.

Two LB isolates, LB01 and LB09, identified as Phb. leiognathi subsp. mandapemensis and Phb. leiognathi, respectively, were selected based on their increased bioluminescence intensity after incubation at 28°C for 48 h (Figures 1A, B). The mean SB of LB01 was 6.04E+06, whereas LB09 exhibited a 1.7 times higher mean SB of 1.04E+7. This indicates that LB09 has a significantly higher bioluminescence capability than LB01. Nevertheless, both strains were studied further.

The growth dynamics of both strains were monitored at two distinct temperatures, 22°C and 30°C, to assess temperature-dependent growth variations (Figures 1C, D). At 22°C, both strains exhibited similar growth phases. The lag phase started <1 h and was immediately followed by the exponential phase. However, at 30°C, LB01 had a <2 h lag phase then followed by a prolonged exponential phase, which continued until approximately 32 h (Figure 1C). On the other hand, LB09 had a 1.5-h lag phase and a shorter exponential phase, which lasted until between approximately 22.5 h (Figure 1D). Both strains exhibited faster growth at the lower temperature.

The effects of GMPs on the bioluminescence intensities of the two Phb. leiognathi strains at 22°C or 30°C were investigated. The SB values were presented in Supplementary Tables S1, S2). The bioluminescence intensities of the two strains exposed to varying GMP concentrations were found heterogeneous (Figures 2, 3).

In the 3.1-day experiment, which was used to assess the immediate effects, both strains displayed early peaked of bioluminescence intensity compared to the control group at 30°C (Figures 2A, B), suggesting an upregulation of QS in response to GMP exposure. Interestingly, a biphasic bioluminescence was observed in LB01 exposed at 0.5% GMP concentration that could reflect adjusted QS in response to the presence of GMPs (Figure 2A). These early bioluminescent responses of the two strains are indication of hormetic effect, where low-dose exposure triggers a stimulatory response. At 22°C, both strains exhibited uniform patterns of bioluminescence when compared to the control group, except for LB09 exposed to 2% GMP concentration (Figures 2C, D).

The 7-day experiment involved monitoring the bioluminescence of both strains at both temperatures, with varying GMP concentrations, which was used to assess the long-term effects of plastic particles (Figure 3). At 30°C, an obvious biphasic bioluminescence phenomenon was observed in LB01 at lower concentrations (0.25% and 0.5%) (Figure 3A). Furthermore, the strain also demonstrated biphasic-dose response, where biphasic bioluminescence observed at lower concentrations and a potential bioluminescence quenching (an inhibitory effect) at higher concentrations (1% and 2%) (Figure 3A). At 22°C, both strains showed complex bioluminescence responses (Figures 3C, D). LB01 exposed to lower concentrations showed delayed bioluminescence peaks (Figure 3C). In contrast, LB09 displayed initial bioluminescence peaks across all GMP concentrations, with a potential biphasic bioluminescence pattern observed specifically at the 0.5% GMP (Figure 3C).

Overall, these results highlight the heterogenous bioluminescence patterns of Phb. leiognathi strains to GMP exposure, with strain-specific responses emerging based in GMP concentration and temperature that lead to differences in their physiological adaptation. Furthermore, the observed biphasic bioluminescence and hormesis underscore the complex interactions between bacterial metabolic processes and MP stressors.

A post-exposure recovery experiment was conducted to determine whether the two strains previously exposed to GMPs for 3.1 days and 7 days could recuperate from the effects of this exposure. The SB values for the first 35 h were presented in Supplementary Tables S3, S4). Following the 3.1-day GMP exposure, the bioluminescence responses of LB01 exposed to all GMP concentrations were comparable to those of the control group, except at 2% GMP, indicating that the strain was able to recuperate from the effects of GMPs at low concentrations (Figure 4A). However, early bioluminescence peaks were observed in LB09 following exposure to 0.5%, 1%, and 2% GMPs, but the bacteria exposed to 0.25% displayed slower peaking of bioluminescence (Figure 4B). At 22°C, early bioluminescence peaks were still observed in LB01 following 3.1-day exposure to GMPs (Figure 4C), while LB09 showed contrasting responses, where the bacteria exposed at higher GMP concentrations have bioluminescence patterns comparable to the control group (Figure 4D).

Following the 7 days GMP exposure, LB01 at 30°C displayed slower peaking of bioluminescence, while exposure to higher GMP concentrations (1% and 2%) remained incomparable with the control group (Figure 5A). Similarly, LB09 exposed to 0.25% GMP concentration at the same temperature also showed slower peaking of bioluminescence, but exposure to 0.5%, 1% and 2% GMP concentrations created initial peaks of bioluminescence (Figure 5B ). At 22°C, the bioluminescence responses of LB01 exposed to lower GMP concentrations were comparable to those of the control group (especially at 0.5% GMP concentration), except at 2%, indicating that the strain was able to recuperate from the effects of GMPs at low concentrations (Figure 5C). However, early bioluminescence peaks were observed in LB09 following exposure to 0.5%, 1%, and 2% GMPs, but the bacteria exposed to lowest GMP concentration also displayed slower peaking of bioluminescence (Figure 5D).

MTT assays were performed to evaluate the effects of GMPs on the cell viability of both strains under different experimental conditions (Figure 6). Then, the mean OD₅₆₀ readings were determined and presented in Supplementary Tables S5, S6). In the 3.1-day experiment at 30°C, LB09 showed no significant decrease in viability compared to the control group (Figure 6A). LB01, where a hormetic effect of low GMP concentrations was previously observed, exhibited with no significant differences in viability at 0.25% and 0.50% GMP concentrations compared to the control groups (Figure 6A). At 22°C, LB01 revealed a significant (p < 0.05) decrease in viability after exposure at all GMP concentrations compared to the control group (Figure 6B). Conversely, LB09 had distinct MTT assays results at the same temperature. The viability decreased significantly (p < 0.05) at low concentrations (0.25% and 0.5%) compared to the control group, but not at high concentrations (1% and 2%), indicating possible adaptive mechanisms (Figure 6B).

Extending the exposure period to 7 days yielded substantial differences (Figures 6C, D). At 30°C, LB01 exhibited a significant (p < 0.05) increase in viability at low concentrations (0.25% and 0.5%) vs. to the control group, but not at high concentrations (1% and 2%) (Figure 6C). In contrast, LB09 demonstrated a significant (p < 0.05) decrease in viability compared to the control group at all concentrations (Figure 6C). At 22°C, LB01 showed a significant (p < 0.05) decrease in viability compared to the control groups at the 2% GMP concentration, whereas LB09 generally exhibited a decrease in viability at the 0.25% GMP concentration compared to control group (Figure 6D). These observations suggest strain-specific differences in bacterial viability to MPs through QS regulation, which influenced by GMP concentration, temperature, and time exposure.

The biofilm formation of the two strains was assessed by crystal violet biofilm formation assays following GMP exposure (Figure 7). The biofilms were quantified and expressed as SBF, which were presented in Supplementary Tables S7, S8).

In the 3.1-day experiment at 30°C, the SBF of LB09 increased significantly (p < 0.05) as the GMP concentration increases compared to the control group (Figure 7A). Similarly, LB01 produced significantly (p < 0.05) high SBF at all GMP concentrations, except at 0.50%, compared to the control group (Figure 7A). On the other hand, at 22°C, the SBF in both strains increased vs. control group following exposure to GMP concentrations, except at 0.50% (Figure 7B). In the 7-day experiment, both strains showed contrasting SBF results at different temperatures (Figures 7C, D). It was observed that the LB01 and LB09 produced significantly (p < 0.05) low SBF as compared to the control group at 30°C and 22°C, respectively (Figures 7C, D). These observations suggest that there were strain-specific responses based on their biofilm formation after exposure to GMPs at lower temperature.