Aspergillus oryzae attenuates quorum sensing -associated virulence factors and biofilm formation in Klebsiella pneumoniae extended-spectrum beta-lactamases

Preparation of Klebsiella pneumoniae extended-spectrum beta-lactamases, Aspergillus oryzae and Caenorhabditis elegans

KP-ESBL (ID.100029) were obtained from the Laboratory of Microbiology, Faculty of Medicine, Brawijaya University, Malang, Indonesia. Luria Broth (LB) medium was used to cultivate the KP-ESBL strain, which was then incubated at 37°C for 16–18 hours. Sterile saline was diluted 100 times after being equalized with the Mc. Farland standard to produce a concentration of 106 CFU/mL. This bacterial suspension was then ready for testing. The fungal strain Aspergillus oryzae was provided by the Indonesian Culture Collection (Ina-CC). Preparation of Aspergillus oryzae begins with sub-culture and preservation of previously isolated Aspergillus oryzae. Sub-cultures were carried out from cryo to Luria Bertani solid medium. The culture was then treated in the form of H₂O₂ and without glucose, then stored at 37°C in an incubator. The nematode Caenorhabditis elegans was maintained on agar medium for nematode growth media (NGM) fed with Eschericia coli OP50. Gravid C. elegans were treated with hypochlorite to synchronize C. elegans culture at the first larval stage. Before being employed for infection, the C. elegans were then reared at 25°C until they reached the young adult stage.

Preparation of Aspergillus oryzae extracellular protein (AOEP)

100 mL of potato dextrose broth (PDB) medium were inoculated with 8-mm (diameter) Aspergillus oryzae mycelium and placed in a 250 mL Erlenmeyer flask containing 2% glucose. The flask was incubated for 72 hours at 27°C in a shaker incubator under static conditions (OD600 = 1.2). The culture was filtered using 0.22-micron filter paper after incubation (Whatman, Sigma Aldrich). As a source of extracellular protein, the supernatant was centrifuged at 12.000 rpm for 15 minutes at 4°C. Ammonium sulfate was used to precipitate extracellular proteins at saturation values of 80%. After one hour of stirring in the ice bath, ammonium sulfate was added to the supernatant. The crude protein extract was centrifuged at 4°C for 15 minutes at 12,000 rpm the next day after being maintained at 4°C overnight. After that, the complete protein precipitate underwent a twenty four hour-dialysis in a 0.01 M phosphate buffer at pH 7 using a 10×-sample volume. After that, the Aspergillus oryzae extracellular protein (AOEP) was prepared for the assay.

Determination of MIC

A growth inhibition test was conducted using microdilution broth. Briefly, fresh cultures were inoculated on LB medium at turbidity equivalent to 0.5 McFarland standard, 500 μL of each bacterial culture were added to a 96-well polystyrene flat-bed microtiter plate. The samples were added to the bacterial suspension in each well at final concentrations ranging from 0 to 150 g/mL. The growth control wells only contained bacteria on LB media and kanamycin as positive control. Double serial dilution of the Aspergillus oryzae extracellular protein (AOEP) tested sample was made starting from the first well by adding 50 μL of the tested sample, dissolved at 150 μg/mL. After incubation at 37°C for 24 hours, the absorbance was measured at 600 nm. The lowest absorbance value of the sample that could reduce more than 90% of the absorbance of the negative control was recorded as the MIC value. All experiments were performed in triplicate. Minimum bacterial concentration (MBC) for each sample was calculated by coating the contents of the first three wells, which showed no visible bacterial growth on the LB plate, and incubated for 24 hours.

Biofilm assay

The test well on a 96-well microplate received a total of 100 μL of Aspergillus oryzae extracellular protein (AOEP) at various concentrations (18.75, 37.5, 75, and 150 μg/mL). The negative control wells received 200 μL of mixed LB media and 1% glucose, while the positive control wells received 64 μg/ml of kanamycin. Each well was then filled with 100 μL of the KP-ESBL suspension. For 24 hours, the microplate was wrapped and kept at 37°C in an incubator. The microplate's contents were taken out the following day, thoroughly cleaned with sterile distilled water three times, and then dried. 200 μL of 0.1 % crystal violet dye was added to each well once the microplate had dried, and it was air dried at room temperature for 15–20 minutes. The microplate's contents were then cleaned with sterile distilled water and dried. After 15 minutes of incubation at room temperature, 200 μL of a 96% ethanol solution were added to each well, and the results were measured at 570 nm with a microplate reader.

Biofilm growth inhibition was calculated using the following formula:

(OD Control = Optical density control negative. OD Test = Optical density test)

Analysis of biofilm structure using SEM

Visual evaluation of AOEP's impact on KP-ESBL morphology was conducted using a scanning electron microscope (SEM, model Zeiss 224 EVO 50 VP, Germany). KP-ESBL bacteria were cultivated in LB broth and incubated for 24 hours in an aerobic environment at 37°C. A 1-mL volume of the bacterial suspension was obtained and treated with AOEP for two hours once it reached around 1×10⁸ CFU/ml. Another 1 mL sample was taken from the culture and left untreated. These two bacterial samples were centrifuged after two hours for three minutes at 1400 rpm, and the pellets were then cleaned twice with 0.1 M phosphate buffer saline (PBS). KP-ESBL cells were exposed to 2.5% glutaraldehyde for two hours at 4°C for the SEM analysis. Samples were exposed to each concentration after fixation for one to two minutes in order to dehydrate them. The samples were then centrifuged at 1400 rpm for 10 min, after which the pellets were re-dispersed in 100% ethanol and air dried. The samples were coated with gold and palladium in an 80:20 ratio prior to examination under SEM at 20 kV. The working magnification was kept at less than 10 mm for better focusing.

Production of cell-free EPS

Cultures of bacterial isolates left overnight were inoculated to 9.5 mL of LB broth along with 0.5 mL of cell lysate and incubated at 30°C for 24 hours. The late-log phase cells attached to the test tube walls were harvested by centrifugation at 8500 rpm for 30 min at 2°C. The filtered supernatant was added with three volumes of cold ethanol and incubated overnight at 2°C to precipitate the released EPS. The precipitated EPS were then collected by centrifugation at 5000×g for 30 min and dissolved in 1 mL of deionized water. Enzyme-free media culture added with PBS served as a control. The bacterial cells were removed, resuspended in sterile PBS, and read at 600 nm. The collected EPS was quantified using the phenol-sulfuric acid method.

C. elegans life span killing assay

Similar to the anti-infection screen, the liquid-based survival test was carried out with a few minor adjustments. A total of 30 young adult C. elegans were used in place of the N2 young adults that received treatment. As a result, the C. elegans were kept at 16°C to create gravid C. elegans, and they were given a hypochlorite treatment to develop eggs. In order to conduct an infection assay, eggs were sown on NGM agar and developed into sterile young adults of C. elegans at 25 °C. Every four hours following infection, both alive and dead C. elegans were counted. Each extract was examined in three wells, each representing about 100 C. elegans. In control wells, dimethyl sulfoxide (DMSO) was used in place of the extract, and Escherichia coli OP50 were fed. To examine the impact of AOEP on KP-ESBL pathogenicity to C. elegans, we performed a slow-killing survival experiment. On a 48-well microplate, KP-ESBL were first cultured for an overnight period at 37°C in the presence of AOEP (18.75, 37.5, 75, and 150 μg/mL). When 100 sterile young adult C. elegans were put into the well, the infection began. KP-ESBL was given DMSO treatment as a negative control in place of AOEP. After 48 hours, the C. elegans that were still alive were counted under microscope with a magnification of 100×.

RT-qPCR analysis

The hot phenol method was used to extract total ribonucleic acid (RNA), where the DNA was removed using TURBO DNA-free (Ambion, Inc.), and the RNA quality was determined using a NanoDrop (ND-1000; Thermo Scientific) and an Agilent 2100 bioanalyzer with a Picochip (Agilent Technologies). After 35 qPCR cycles, the absence of contaminating DNA was determined by the absence of amplification products. A 1 μg of RNA, random hexamer primers (0.2 μg/L), and M-MulV-RT (20 U/L, Moloney murine leukemia virus reverse transcriptase; Thermo Fisher Scientific) were used to synthesize cDNA. Specific primers for mrkA 5′-CGGTAAAGTTACCGACGTATCTTGTACTG-3′, and wzI 5′-GCTTAYGCRGCYGGGTTAGTRGT-3′ designed with the Primer3Plus software (Primer3Plus is an open alternative). A master mix of the following components was prepared for light cycler reactions: 3.0 mL PCR-quality water, 1.0 μL (10 M), 10 μL 2× SYBR Green I Master Mix, 10 μL reverse primer, and 5.0 μL cDNA (50–100 ng). A multi-well plate was sealed with sealing foil, centrifuged for two minutes at 1500 g, and loaded into the LightCycler 480 instrument (Roche). For each sample examined, amplification was carried out in triplicate wells. All reactions had control reactions with no template (water) and minus-reverse transcriptase (RNA). Cycling conditions were as follows: denaturation (95°C for 10 minutes); amplification and quantification repeated for 45 cycles (95°C for 10 seconds, 57°C for 20 seconds, 72°C for 30 seconds with a single fluorescence measurement); melting curve (95°C for 10 seconds, 65°C for one minute with continuous fluorescence measurement at 97°C); and finally, a cooling step at 40°C for 10 seconds. After each run, a melting curve analysis was performed to confirm the specificity of the primers. For normalization, 16S rRNA was used as a reference gene, and relative gene expression was calculated using the 2Ct method.

AOEP inhibited the growth of KP-ESBL

The antimicrobial activity of AOEP was quantitatively assessed by measuring the turbidity at a wavelength of 600 nm. The results in Figure 1 represent crude proteins’ MIC and MBC values in various concentrations with kanamycin as positive control. The concentration of AOEP, which could inhibit > 90% of the bacterial population, represented MIC and was 300 μg/mL. The concentration used in the growth inhibition test of antibiofilm activity was sub-MIC, namely at 1/8 and 1/16 × MIC. This study used a 64-μg/mL dose of kanamycin as a positive control.

Figure 1. Minimal inhibitory concentration (MIC) of Aspergillus oryzae extracellular protein (AOEP) against Klebsiella pneumonia extended-spectrum beta-lactamases (KP-ESBL).

MIC at a concentration of 300 μg/mL was 92.74% (bold). Kanamycin as a positive control, was only able to inhibit KP-ESBL 78.51%. The negative control of KP-ESBL without AOEP exposure was 0%. Bars indicate the standard error, and the sign (*) above the bars indicates a significant difference (p < 0.05).

The highest concentration used for the AOEP inhibition test against KP-ESBL biofilms was 150 μg/mL, which was the MIC (p-value < 0.05). The Tukey Post Hoc test showed that there were significant differences between the overall treatment group and the negative control. The linear regression test results showed a R-value of 0.797, reflecting that AOEP could inhibit the growth of KP-ESBL in a dose-dependent manner. Furthermore, measurement of the inhibitory ability of biofilms and virulence factors used sub-MIC concentrations of 1/8 and 1/16 MIC, there were 18.75, 37.5, 75, and 150 μg/mL.

AOEP inhibited the formation of KP-ESBL biofilms at MIC

The microdilution method was used to test the inhibitory activity of the AOEP biofilm against KP-ESBL. Figure 2 displays the AOEP biofilm's inhibitory efficacy against KP-ESBL.

Figure 2. Inhibition of Aspergillus oryzae extracellular protein (AOEP) against Klebsiella pneumonia extended-spectrum beta-lactamases (KP-ESBL) biofilms.

Inhibitory effect of AOEP on biofilms after co-incubation for 24 h with different concentrations of AOEP. The concentration of AOEP is given relative to MIC KP-ESBL. The AOEP biofilm inhibition concentrations of 18.75, 37.5, 75, and 150 μg/mL were 32.94%, 39.17%, 68.14%, and 72.18%, respectively. Bars indicate the standard error, and the sign (*) above the bars indicates a significant difference (p < 0.05).

Figure 3. Reduction of Aspergillus oryzae extracellular protein (AOEP) against cell-free Klebsiella pneumonia extended-spectrum beta-lactamases (KP-ESBL) exopolysaccharide.

Inhibitory effect of AOEP on cell-free exopolysaccharide KP-ESBL after being incubated together for 24 hours with different concentrations: 18.75, 37.5, 75, and 150 g/mL. Bars indicate the standard error and the sign (*) above the bars indicates a significant difference (p < 0.05). KP-ESBL

AOEP was administered relative to the MIC. The MIC value of the K. pneumoniae strain was 300 μg/mL, so the highest concentrations for the anti-biofilm test were 1/8, 1/16 MIC. The biofilm inhibition value [100-(sample ABS/control ABS × 100)] for each AOEP concentration can be seen in Figure 2. In the crystal violet staining assay, KP-ESBL biofilms were significantly inhibited at concentrations of 1/4 × MIC (75 μg/mL) and 1/2 × MIC (150 μg/mL) (p < 0.05). Interestingly, AOEP inhibited biofilm formation at concentrations below the MIC. The ability of AOEP to inhibit biofilm formation in KP-ESBL exceeded the ability of the kanamycin (69.46 μg/mL and 37.9 μg/mL). The positive control had a biofilm inhibitory value of 37%, which was lower than the AOEP biofilm inhibitory level of 150 and 75 μg/mL. The negative control (KP-ESBL bacteria without AOEP exposure) showed the lowest biofilm inhibition value (0), which indicated that biofilm production was not inhibited at all. When administering the four concentrations of AOEP, the resulting OD value decreased significantly as the dose increased when compared with the OD of the negative control. This indicates AOEP inhibition of the KP-ESBL biofilm. Tukey test results indicated that there were significant differences between the overall treatment group against the negative control. The linear regression test results show that the R-value (0.957) that represented AOEP could inhibit the growth of KP-ESBL biofilm in a dose-dependent manner.

AOEP reduced cell-free exopolysaccharide of KP-ESBL

The assay was performed to test the ability of AOEP to reduce cell-free exopolysaccharide KP-ESBL.

KP-ESBL treated with AOEP 150 μg/mL could reduce the bond matrix with Congo Red dye by as much as 49% after staining and assessment with a spectrophotometer. As the dose of AOEP was reduced (75 μg/mL, 37.5 μg/mL, 18.75 μg/mL), its inhibition ability decreased (42%, 38%, 37%). Cell-free EPS might be reduced by 19% using AOEP 150 μg/mL and it surpassed the kanamycin (30 %). There was a significant difference, according to the one way ANOVA test (p-value < 0.05). The results of the Tukey Post Hoc test revealed that the overall treatment group and the unfavorable control group differed significantly. The findings of the linear regression test indicated that AOEP might inhibit the cell-free EPS KP-ESBL in a dose-dependent manner, and the R-value for this test was 0.896.

AOEP disrupted fimbriae through downregulation of the merized fimbrial shaft (mrkA) genes

The delta-delta Ct method (2 DDCt) was used to quantify RT-qPCR results. Results are represented as “Target/adh3 fold change.” The results of gene expression analysis via RT-qPCR (Figure 4) showthat AOEP downregulated the gene expression for fimbriae mrkA, which acts as an adhesion molecule. Meanwhile, capsular EPS as measured by the wzI gene expression was increased.

Figure 4. Effect of Aspergillus oryzae extracellular protein (AOEP) on the gene expression coding for the fimbriae and capsule of Klebsiella pneumonia extended-spectrum beta-lactamases (KP-ESBL).

The expression of these two genes was measured in response to AOEP: mrkA (type 3 fimbrial shaft) and wzI (surface assembly of capsule). The expression of the adh3 house-keeping gene was used as an internal control for each sample. The concentration of AOEP treatment was 150 μg/mL, while the control group was KP-ESBL without AOEP exposure.

AOEP disturbed the biofilm structure of KP-ESBL on observation under SEM

The impact of AOEP on cellular alterations was examined using SEM analysis. To supplement the information of the quantity of the biofilm, observation of the architecture of the biofilm mass using SEM was conducted. The impact of AOEP on the KP-ESBL biofilm structure was demonstrated by SEM data (Figure 5).

Figure 5. Scanning electron microscope (SEM) micrograph image of Klebsiella pneumonia extended-spectrum beta-lactamases (KP-ESBL) cells exposed to Aspergillus oryzae extracellular protein (AOEP) sub-minimum inhibitory concentration (MIC, 5000× magnification left, 10,000× magnification right).

K. pneumoniae biofilms grew after incubation for 24 hours. AOEP was added with different concentrations: 150 μg/mL (B), 75 μg/mL (C), 37.5 μg/mL (D), 18, 75 μg/Ml (E) Positive control with the addition of the Kanamycin (F).

The negative control group showed bacterial colonies along with thick biofilms evenly distributed on the adhesion surface (Figure 5A). This was different from when the bacteria were treated with the 150 μg/mL AOEP (Figure 5B), the cells failed to aggregate, and there was a highly significant decrease in biofilm mass. In this group, the bacterial colonies were separated and became planktonic bacteria, and the adhesion surface was free of bacterial biofilms. Biofilm mass was also decreased in the AOEP 75 μg/mL (C) group, while the positive control group (Kanamycin, F) showed partial inhibition of the KP-ESBL biofilm. It can be seen that the biofilm structure of KP-ESBL was impaired due to the addition of AOEP when compared to the control. The control group was KP-ESBL which was not exposed to AOEP, as shown in Figure 5A. When bacteria stick together, the attachment of bacteria is more clearly facilitated by a thick mass of biofilm surrounding the bacterial colony. In this group, the biofilm appeared to cover all bacterial colonies on the surface of the adhesion medium. This appearance differed significantly from the group treated with 150 μg/mL of AOEP (Figure 5B) provides a clearer picture of the dispersal in the bacterial colonies treated with AOEP reflecting the impair the biofilm. The inhibition of biofilm mass formation in the group exposed to AOEP at a dose of 75 μg/mL (Figure 5C) also shows that the bacterial colony dispersed. However, the number of bacteria was higher than for a concentration of 150 μg/mL. At an AOEP dose of 37.5 μg/mL (Figure 5D), it was seen that some bacteria were separated, and some bacteria were attached (left). At 10000× magnification, a biofilm mass began to surround the bacteria and facilitated adhesion between bacteria and the adhesion medium.

Meanwhile, at the lowest concentration of AOEP, a dose of 18.75 μg/mL (Figure 5E), the presence of a thick biofilm was seen that matched the negative control. Interestingly, the sub-MIC ability of AOEP to reduce biofilm mass formation produced stronger effect than the kanamycin (Figure 5F). Overall, SEM results showed the highest reduction in biofilm mass formation occurred with a treatment of AOEP 150 μg/mL, which had a stronger effect than the kanamycin. These results indicate that AOEP can be used as a candidate antibiofilm agent at concentrations lower than MIC, especially against biofilm formation by KP-ESBL.

AOEP used a competitive antagonistic mechanism by producing QS-like Gram Negative bacteria molecule to interfere with KP-ESBL virulence

A liquid chromatography-mass spectrometry LC-MS/MS study against AOEP was carried out in order to identify the A. oryzae molecule that contributes to KP-ESBL virulence and biofilm suppression. The results were displayed as a chromatogram, which showed the peak height and molecular weight of the sample substance. Figure 8 and Table 3 show the outcomes of the LC-MS/MS study.

Figure 8. Ion-extracted liquid chromatography-mass spectrometry (LC-MS/MS) chromatogram of a mixture of standard AHL and AQ molecules showing separation.

In order (from top to bottom), 3-oxo-C6-HSL, 3-OH-C6-HSL, C10-HSL, NHQ, NQNO, 3-OH-C12-HSL.

The chromatograms showed a number of substances with various peaks and molecular weights. Six compounds had prominent and high peaks (Figure 8). Based on the precursor ion (m/z), ion product (m/z), cone voltage, and impact energy, the six peaks were identified. The six peaks contained substances with properties resembling those of the QS substance K. pneumoniae. The six substances were NHQ, NQNO, 3-OH-C12-HSL, 3-oxo-C6-HSL, and C10-HSL. The six compounds were found to match the typical precursor parameters 3-oxo-C6-HSL, 3-OH-C6-HSL, C10-HSL, NHQ, NQNO, and 3-OH-C12-HSL (see Table 1).

Underlying data

Figshare: Aspergillus oryzae attenuates quorum sensing -associated virulence factors and biofilm formation in Klebsiella pneumoniae extended-spectrum beta-lactamases raw data, https://doi.org/10.6084/m9.figshare.20290929.²⁸

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).