Interactions of semiconductor Cd-based quantum dots and Cd²⁺ with gut bacteria isolated from wild Salmo trutta fry

Materials

In this study commercially available semiconductor Cd-based CdSe/Zns core/shell QDs (cat. No. A10200; Life Technologies, Carlsbad, CA, USA) were used. The QDs were coated with a polymer layer with carboxylic acid (−COOH) side groups that allow facile dispersion of QDs and got a negative surface charge in aqueous solutions with retention of their optical properties. The certified standard Cd²⁺ (1,000 mg/L) in HNO₃ (0.5 mol/L, Certipur® solution) was purchased from Merck (Darmstadt, Germany).

Spectroscopic measurements of QDs

The stability of the spectral properties of CdSe/ZnS-COOH QDs were measured in phosphate KH₂PO₄ –NaOH buffer solution (PBS) (0.05 M) with different pH values (pH 5.0 and pH 7.0) in the dark at room temperature (about 18 °C). Some samples at pH 7.0 were constantly illuminated over 48 h using a white fluorescent lamp (11W/827; Osram Duluxstar, Taiwan, China) from a distance of about 25 cm. The pH 5.0 samples were made from PBS pH 7.0 acidified with HCl (12 M). The concentration of QDs in the stock solution was 8 µM. The 1 ml volumes of QDs samples were prepared by diluting the stock solutions with PBS to a main final concentration of 4 nm. The measurements of the spectra of the QDs samples were made in a 10 × 4 mm quartz cuvette (Hellma, Jena, Germany) 1 h after the beginning of the experiments, the during 24 h and after 48 h. The absorbance spectra were measured using an AvaSpec-2048 fiber optic (Avantes, Apeldoorn, The Netherlands) absorption spectrometer, while a LS55 spectrofluorimeter (PerkinElmer, Waltham, MA, USA) was used to register PL spectra. An excitation wavelength for PL was set at 405 nm, the excitation slit was 10.0 nm, and the emission slit was 2.5 nm. The CdSe/ZnS-COOH QDs possessed a strong PL in the red spectral region, with a PL peak of 620 nm, and the initial full width at half maximum (FWHM) was about 25 nm in the PBS buffer solutions with pH 7.0 (Fig. S1). OriginPro9 (OriginLab, Northampton, MA, USA) software was used for data analysis.

Isolation and identification of wild Salmo trutta fry gut bacteria

Wild Salmo trutta fry specimens were collected from the Kena River (54.650756, 25.633395) by electrofishing in early autumn 2020. The water temperature in the Kena River ranges from 2.5 °C to 18 °C. The collected fish specimens were euthanized by a blow to the head following international, national, and institutional guidelines for the care and use of animals (Directive 2010/63/EU; LT 61-13-005). Fish specimens were aseptically dissected and the gut contents were squeezed out. Serial dilutions of homogenates were prepared in sterile PBS (pH 7.0, Oxoid Ltd, Hampshire, UK) (up to 10⁻⁶), from which 100 μL aliquots were spread onto Tryptone Soya Agar (TSA, Oxoid Ltd, Hampshire, UK) plates. The plates were then incubated at 20 °C for 48–72 h. Twenty-six morphologically different colonies were randomly picked and re-streaked on TSA plates. Bacterial genomic DNA extraction and molecular identification of isolates were performed as previously described (Skrodenyte-Arbaciauskiene et al., 2012). Polymerase chain reaction (PCR) products 1,500 bp were sequenced using an automated ABI 310 DNA Sequencer and the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Waltham, MA, USA). The NCBI Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/) and Ribosomal database project classifier (RDP; http://rdp.cme.msu.edu/) were used to assess similarity between the obtained 16S rRNA gene 1,500 bp sequences and sequences contained within the nucleotide database.

The 16S rRNA gene sequences were deposited in the DDBJ/EMBL/GenBank databases under the following accession numbers: MW441229–MW441233; OL739143–OL739153.

Antibacterial assay by disc-diffusion method

The antibacterial effect was studied by disk-diffusion assay according to Mir et al. (2018) and Travlou et al. (2018). The gut bacterial cultures were grown overnight on Tryptone Soya Broth (TSB, Oxoid Ltd, Hampshire, UK) until reaching the optical density of 1.0 ± 0.1 at 600 nm, corresponding to 10⁸ colony forming units (CFU) per ml. A 100 mL aliquot of the bacterial culture was evenly spread on a TSA agar plate. Sterile paper discs (6 mM diameter, Whatman, Maidstone, UK) impregnated with 10 µL of Cd²⁺ (at the concentration 0.09–3.56 mM or 10–400 mg/L) and Cd-based QDs suspensions (at the concentration of 4 nm) (pH 7.0) were placed on these bacterial spread plates (4–5 paper disk replicates of the same concentration). QDs and Cd²⁺ samples were prepared by diluting stock solutions with PBS (pH 7.0) to the final concentration. Blank samples were sterile discs impregnated with sterile PBS solution (pH 7.0). The plates were incubated at 15 °C and 25 °C for 48 h in natural light (combined day/night) and in the dark. The experiment was repeated with Cd-based QDs at pH 5.0. The Cd-based QDs samples were prepared by diluting the stock solution with PBS (pH 5.0) to the final concentration of 4 nm. PBS, TSA, and TSB were acidified with HCl to pH 5.0. HCl was used because parietal cells in the mucosa secrete HCl into the fish’s stomach. After incubation, the Petri dishes were photographed using a smart-phone camera. Images were produced under UV light. The diameters of the growth inhibition zone were measured using the ImageJ software image analyzer and expressed as mm (mean ± SD). MIC values were assessed by determining the lowest concentration causing bacterial growth inhibition.

Interactions of Cd-based QDs with bacteria: spectroscopic measurements after 9 h exposure

Ethics approval

All animal work was conducted on public land and waterways, it did not involve protected, threatened or endangered species, and complied with relevant national and international guidelines and legislation. A permit to perform sampling of Salmo trutta fry specimens in the Kena River, Permit No. 025, was issued in 2020 to the Nature Research Centre by the Environmental Protection Agency under the Ministry of the Environment of the Republic of Lithuania. All applicable international, national and institutional guidelines for the care and use of animals were followed (Directive 2010/63/EU; LT 61-13-005).

Statistical analysis

For statistical evaluation, data on the MIC zone were first checked for normal distribution using Kolmogorov–Smirnov and Shapiro–Wilk tests. A two-way ANOVA (F-statistics, p < 0.05) followed by a Post-hoc Tukey’s HSD test was used for data. STATISTICA (7.0 Software, Inc., Exton, PA, USA) was used to calculate the statistics.

The spectroscopic properties of QDs

The spectroscopic properties of QDs were investigated to determine the structural stability of the QDs under the applied experimental conditions. The initial optical density of CdSe/ZnS-COOH QDs was found to be higher at pH 5.0 than at pH 7.0 (Fig. 1A). In contrast, the initial PL intensity of QDs was lower at pH 5.0 and higher at pH 7.0 (Fig. 1B). The PL spectrum of QDs had a peak at about 620 nm in PBS with pH 7.0, but a bathochromic shift of about two nanometres was observed in the more acidic solution (Fig. 1A, insert). Thus, as expected, the CdSe/ZnS-COOH QDs demonstrated better initial spectroscopic properties (a higher intensity of PL and a bigger quantum yield) in PBS with pH 7.0 than in pH 5.0 1 h after preparation of samples. The decreased PL intensity with the bathochromic shift in the acidic media can be associated with the tendency towards stronger aggregation, which occurs in QDs samples with lower pH.

After 1 day in the dark at room temperature, the PL intensity of Cd-based QDs decreased by about 25% from the initial value in the samples with pH 7.0 and by about 60% with pH 5.0 (Fig. 2A). The peak PL intensities of all QDs samples decreased over 2 days, though a faster decrease was observed in solutions with pH 5.0. During this period of time, not only was a decrease in intensity of the PL band observed in the QDs samples, but also a progressing bathochromic shift under illumination at pH 7.0 for 48 h (Fig. 2B). However, this shift could not reach the degree that was registered immediately in more acidic media. On the other hand, the spectral position of the PL peak at pH 5.0 remained stable during the experiment time. Thus, the difference in pH of the samples caused stronger effects on the optical properties of CdSe/ZnS-COOH QDs than environmental lighting conditions over 48 h, although it seems that the same mechanisms of QDs surface modification were predominant in both cases.

Bacteria identification

The 26 bacterial isolates from gut of wild Salmo trutta fry were identified as Gram-positive (belonged to Carnobacterium, Listeria and Microbacterium genera) and Gram-negative (belonged to Aeromonas, Buttiauxella, Serratia and Shewanella genera) bacteria. Sequences of isolated strains AL13; AL20; ST8; ST9; ST16 closely related to Aeromonas salmonicida (sequences similarity 99%); AL10; ST3; ST4; ST7; ST12; ST18—to Aeromonas sobria (similarity 99%), AL2—to Aeromonas popoffii (similarity 99%), AL8; ST11; ST15—to Aeromonas spp. (similarity 100%), AL18; ST1; ST10; ST13; ST14—to Shewanella putrefaciens (similarity 99%), ST6; ST17—to Carnobacterium maltaromaticum (similarity 100%), ST19—to Listeria sp. (similarity 99%), ST32—to Buttiauxella sp. (similarity 99%) and ST33—to Serratia sp. (similarity 98%) and ST5—to Microbacterium sp. (similarity 99%) (Table S1).

Antibacterial efficacy of Cd-based QDs and Cd²⁺

Antibacterial activity of Cd-based QDs and Cd²⁺ was tested on the 26 gut bacteria isolated from wild Salmo trutta fry in disk-diffusion assays. Generally, antimicrobial agent diffuses into the agar, this inhibiting germination and growth of the test microorganism and resulting in hollow zones forming around the discs (Balouiri, Sadiki & Ibnsouda, 2016). The agar disc-diffusion method yields qualitative interpretive category results (susceptible or resistant) and a quantitative result (zone of inhibition) (Espinel-Ingroff, 2007). Thus, the more susceptible to the antimicrobial activity of the compound a strain is, the larger the diameter of the inhibition zone is.

Under the experimental conditions (pH 7.0, 15 °C and 25 °C, 48 h, in light and in dark), the Cd-based QDs at the concentration of 4 nm did not show antibacterial efficacy on any of the 26 tested Gram-positive and Gram-negative gut bacteria. The experiments on the gut bacteria were repeated with Cd-based QDs at pH 5.0. The agar disc-diffusion assay showed that in the acidic medium, at 15 °C and 25 °C, Cd-based QDs (4 nm) also did not exert a growth inhibition effect on any of the 26 isolated gut bacteria. The results of these studies performed on the Gram-positive (Listeria sp. ST19, C. maltaromaticum ST17, Microbacterium sp. ST5) and Gram-negative (Serratia sp. ST33, Aeromonas sp. ST11, S. putrefaciens ST10) bacteria are presented in Figs. 3A and 3B.

As can be seen in Fig. 4 and Table S1, Cd²⁺ displayed antimicrobial activities on both Gram-positive and Gram-negative gut bacteria. The resistance abilities of the isolated gut bacteria to Cd were found to widely vary, i.e., the established MIC values of Cd²⁺ ranged from 0.09 to 3.11 mM (Fig. 4), and the diameter values of the growth inhibition zones at these concentrations varied from 6.2 to 10.1 mm (Table S1). The obtained data showed that temperature was not a significant factor influencing the MIC value of Cd in 73% of the isolates (19 out of 26), (Fig. 4). However, the temperature was a significant factor affecting the size of the inhibition zone for some isolates at the same MIC of Cd (Table S1). The inhibition zones of six isolates Aeromonas sp. (AL8), A. salmonicida (ST9, ST16), Buttiauxella sp. (ST32), S. putrefaciens (ST 13) and Microbacterium sp. (ST5) were significantly larger (F_1.6 = 8.989, p = 0.024, F_1.6 = 16.35, p = 0.007, F_1.6 = 7.049, p = 0.038, F_1.6 = 8.989, p = 0.024, F_1.6 = 23.11, p = 0.003, F_1.6 = 18.24, p = 0.005, and F_1.6 = 16.89, p = 0.006, respectively) at 15 °C than at 25 °C, and four isolate’s A. sobria (ST3, ST7), S. putrefaciens (AL18, ST10) were significantly larger (F_1.6 = 95.53, p < 0.001, F_1.6 = 34.35, p = 0.001, F_1.6 = 8.218, p = 0.029, and F_1.6 = 31.40, p = 0.001, respectively) at 25 °C than those at 15 °C (Table S1).

Of the bacteria tested, Gram-positive Listeria sp. ST19 was found to be the most sensitive to Cd²⁺ exposure, because the MIC values of Cd were 0.18 and 0.09 mM at 15 °C and 25 °C temperature respectively. The obtained results indicated that the inhibition zones of the Listeria sp. ST19 strain increased with the increasing Cd ions concentrations at 15 °C (r = 0.832) and 25 °C (r = 0.896) temperatures (Figs. 5A and S2A). The MIC value of Cd for the Gram-positive C. maltaromaticum ST17 strain was the same (0.27 mM) in both tested temperatures and the inhibition zones at this concentration did not statistically differ (F_1.6 = 0.009, p = 0.926). In addition, it was noted that the size of the hollow zones around the disc exhibited increasing trend (r = 0.988 at 15 °C temperature and r = 0.978 at 25 °C temperature) with increasing Cd²⁺ concentration. However, a significant difference between the inhibition zones was not observed between the two (15 °C and 25 °C) tested temperatures (F_4.30 = 0.281, p = 0.888) (Figs. 5B and S2B). The MIC values of Cd ions for the C. maltaromaticum ST6 strain were higher (2 and 1.7 times at 15 °C and 25 °C, respectively) than those of the ST17 strain (Table S1). The Gram-positive Microbacterium sp. ST5 exhibited the greatest resistance to Cd ions impact (MIC values were 3.11 mM at 15 °C and 3.29 mM at 25 °C). A significant difference between the inhibition zones was found between the two tested temperatures (F_4.30 = 461.36, p < 0.001) (Figs. 5C and S2C). The high MIC exhibited by ST5 against Cd²⁺ suggests that this strain is likely readily able to survive in an environment with high levels of cadmium contamination.

The MIC values of Cd²⁺ for Gram-negative bacteria (18 out of 22 isolates) ranged from 0.44 to 0.71 mM and did not significantly differ at the tested temperatures (p > 0.05) (Table S1). According to the MIC values of Cd, the most sensitive among the tested Gram-negative bacteria were the Serratia sp. ST33 and Aeromonas spp. (ST11, ST15) strains (Fig. 4). A significant difference between the inhibition zones of Serratia sp. ST33 was found between the two tested temperatures (F_3.24 = 293.41, p < 0.001) (Figs. 5D and S3A). The MIC values of Cd²⁺ for Aeromonas sp. ST11 did not significantly differ between the tested temperatures (F_3.30 = 1.88, p = 0.156) (Figs. 5E and S3B). The MIC value of Cd²⁺ for the S. putrefaciens ST10 strain was the same (0.53 mM) in both tested temperatures, but the diameter of the inhibition zone at this concentration statistically significant increased (F_1.6 = 31.40, p = 0.001) at 25 °C compared to 15 °C (Figs. 5F and S3C).

Interactions of Cd-based QDs with bacteria after 9 h exposure in light

To study the interactions of Cd-based QDs with bacteria after 9 h exposure at pH 5.0, spectroscopic measurements were performed on three Gram-positive (Listeria sp. ST19; C. maltaromaticum ST19; Microbacterium sp. ST5) and three Gram-negative (Serratia sp. ST33; Aeromonas sp. ST11; S. putrefaciens ST10) bacteria (Fig. 6). Initially, the autofluorescence signals of the samples with Gram-negative bacteria were found to be stronger. There were certain specific differences observed between the strains of bacteria in both groups. The spectral position and variation of the PL bands being observed in the red spectral region at the two excitation wavelengths 400 nm (Fig. 6A) and 440 nm (Fig. 6B) can be explained by the presence of different endogenous metalloporphyrins in the bacterial samples. Thus, the signal was strongest in the case of the S. putrefaciens ST10 from Gram-negative and Microbacterium sp. ST5 from Gram-positive strains, while in the case of ST19, the formation of metal-free porphyrins was observed as well.

The PL intensity of the Cd-based QDs in the bacterial culture with supernatant (PBS, pH 5.0) after 9 h in natural light was the higher with Gram-positive bacteria than in samples with Gram-negative bacteria (Fig. 7A). After resuspension in fresh PBS (pH 5.0), the PL intensities of QDs decreased in all samples, but relatively the biggest decrease was registered in samples with Microbacterium sp. ST5 bacterial culture (Fig. 7B). In terms of intensities in bacterial cultures with supernatant, the fluorescence spectra of the supernatant showed the relatively highest PL intensities of QDs in Microbacterium sp. ST5 and Serratia sp. ST33 and the relatively smallest in Listeria sp. ST19 and Aeromonas sp. ST11 (Fig. 7C).

Bacterial colony count results after exposure to Cd-based QDs in PBS (pH 5.0) are presented in Table 1. Experiments were in clear agreement with the data of the disc-diffusion assay and demonstrated that the Cd-based QDs (at the concentration of 4 nm) had insignificant effects on the viability of bacteria after 9 h exposure at 25 °C, pH 5.0 in natural light. The number of bacteria in the control samples did not significantly differ (F_5.24 = 0.362, p = 0.870) from the bacteria numbers after exposure with QDs in a liquid medium (Table 1).