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Low-dose zinc oxide nanoparticles trigger the growth and biofilm formation of Pseudomonas aeruginosa: a hormetic response

BMC Microbiology volume 24, Article number: 290 (2024) Cite this article

Abstract

Introduction

Hormesis describes an inverse dose-response relationship, whereby a high dose of a toxic compound is inhibitory, and a low dose is stimulatory. This study explores the hormetic response of low concentrations of zinc oxide nanoparticles (ZnO NPs) toward Pseudomonas aeruginosa.

Method

Samples of P. aeruginosa, i.e. the reference strain, ATCC 27,853, together with six strains recovered from patients with cystic fibrosis, were exposed to ten decreasing ZnO NPs doses (0.78–400 µg/mL). The ZnO NPs were manufactured from Peganum harmala using a chemical green synthesis approach, and their properties were verified utilizing X-ray diffraction and scanning electron microscopy. A microtiter plate technique was employed to investigate the impact of ZnO NPs on the growth, biofilm formation and metabolic activity of P. aeruginosa. Real-time polymerase chain reactions were performed to determine the effect of ZnO NPs on the expression of seven biofilm-encoding genes.

Result

The ZnO NPs demonstrated concentration-dependent bactericidal and antibiofilm efficiency at concentrations of 100–400 µg/mL. However, growth was significantly stimulated at ZnO NPs concentration of 25 µg/mL (ATCC 27853, Pa 3 and Pa 4) and at 12.5 µg/mL and 6.25 µg/mL (ATCC 27853, Pa 2, Pa 4 and Pa 5). No significant positive growth was detected at dilutions < 6.25 µg/mL. similarly, biofilm formation was stimulated at concentration of 12.5 µg/mL (ATCC 27853 and Pa 1) and at 6.25 µg/mL (Pa 4). At concentration of 12.5 µg/mL, ZnO NPs upregulated the expression of LasB ( ATCC 27853, Pa 1 and Pa 4) and LasR and LasI (ATCC 27853 and Pa 1) as well as RhII expression (ATCC 27853, Pa 2 and Pa 4).

Conclusion

When exposed to low ZnO NPs concentrations, P. aeruginosa behaves in a hormetic manner, undergoing positive growth and biofilm formation. These results highlight the importance of understanding the response of P. aeruginosa following exposure to low ZnO NPs concentrations.

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Introduction

Pseudomonas aeruginosa is a Gram-negative bacterium which is commonly found in a wide variety of environments, including soil and oceans [1]. It is also a clinically significant, opportunistic pathogen which causes morbidity and mortality, especially in individuals with immune system compromise [2]. Patients with cystic fibrosis (CF) are among those who are most vulnerable to P. aeruginosa, which infects the lungs. Indeed, by the time CF sufferers reach adolescence, the lungs of the majority of such patients have become chronically infected with P. aeruginosa. The bacterium is the leading cause of death in this population [3, 4].

The formation of biofilms is a key factor in antibiotic resistance demonstrated by P. aeruginosa [5]. In order for biofilms to form, individual P. aeruginosa bacteria need to communicate using a process termed quorum sensing (QS). As well as enabling biofilm formation, QS regulates the virulence and metabolism of bacteria and planktonic cells [6, 7]. Anti-microbial treatments are often ineffective in resolving P. aeruginosa infections, as they are unable to penetrate the biofilm. They therefore only access the biofilm’s uppermost layers, leaving the deeper bacterial cells in the biofilm matrix untouched [8]. In response to the significant health threat posed by the global rise in antibiotic resistance [9], there is an increasing demand for alternative, non-antibiotic strategies to be devised in order to tackle antibiotic-resistant microbial infections. One potential strategy is to use nanoparticles (NPs), which are readily synthesized and, inherently, have large surface area: volume ratios. These characteristics make them suitable candidates for development into anti-biofilm agents [10].

According to the Food and Drug Administration’s categorization criteria, zinc oxide (ZnO) is generally recognized as safe [11]. Compared to large-sized particles of ZnO, ZnO NPs possess greater antimicrobial activity. This is attributed to their small size and high surface area: volume ratio, which facilitates their interaction with bacteria [12]. Researchers have reported that ZnO NPs are effective disinfectants, and they have been recommended for the treatment of nosocomial infections [13]. It is widely accepted that ZnO NPs has anti-bacterial attributes, which has an inverse correlation with their size and a direct proportion to their concentration [14]. ZnO NPs is effective for their anti-bacterial purpose due to its small sized particles capable of penetrating the membranes of the bacteria [15]. Studies were conducted to determine the correlation between antibacterial activities and particle size. The findings have shown that small NPs with large surface area has higher ability to promote the growth of bacteria [16,17,18]. Smaller particles with antibacterial activities are linked to the rising oxygen species concentration on the surface of the particle as a result of a larger surface area [19].

Despite the fact that ZnO NPs have promising anti-bacterial properties, their safety for use in humans has yet to be established [20]. Their application in a chemotherapeutic role has been limited by their cytotoxic effects. With ZnO NPs doses of 25 µg/mL and above, human lung cell viability was marked diminished [21]. Less toxic effects were seen following exposure to more dilute ZnO NPs concentrations [17]. It is therefore important to study the response of P. aeruginosa to ZnO NPs administered in low concentrations. The aim of the current study, therefore, is to investigate the effects of low concentrations of ZnO NPs on the growth of P. aeruginosa, and on its ability to form a biofilm.

In the current study, the seed extract of Peganum harmala is used as a reducing compound for the green synthesis of ZnO NPs. A member of the Zygophyllaceae family, the medicinal properties of P. harmala have been well-documented. The plant has black, spherical seeds which have a diameter of approximately 5 mm. They are frequently used as a spice in cooking, and have a nutty scent and sharp taste. Conditions which have been treated using P. harmala seeds include lumbago, asthma, colic, jaundice and the promotion of menstrual bleeding [22, 23]. P. harmala has also been attributed with activity against bacteria, fungi and viruses [23, 24]. NPs produced using an extract from this plant have been demonstrated to impact bacterial growth and biofilm formation in a range of Gram-negative organisms [25]. As far as the authors are aware, this is the initial study to investigate the response of clinical P. aeruginosa isolates, obtained from patients with CF, to various ZnO NPs concentrations biosynthesized using P. harmala seeds extract.

Methods

Green synthesis of zinc oxide nanoparticles

Aqueous P. harmala seed extract preparation

Complete plants of P. harmala were harvested in September, 2023, from a farm owned by the lead researcher in the Abbin area of Ajloun Governorate/Jordan. A comparison of the seeds from these plants was made against samples of P. harmala seeds from the herbarium in the Faculty of Agriculture at Jerash University in Jordan. A token sample (AM/2023/01/002) was then placed in storage in the Plant Protection Department’s herbarium at the same university. Institutional, regional and national legislation was adhered to with respect to gathering the P. harmala plants.

The P. harmala seeds were air dried in ambient conditions, rinsed in double-distilled water (Sigma-Aldrich), and then machine-ground to create a coarse-grade powder. 5 g powder, together with 50 ml double-distilled water, was warmed to 75° C for an hour and a half prior to two filtration passes through Whatman paper. Microfiltration was then carried out using a syringe membrane filter (0.22 mm) at 25 °C. This process produced a transparent aqueous extract (pH, 6.4). Fourier-Transform Infrared Spectroscopy (FT-IR) of P. harmala seeds extract was conducted in Hashemite University’s chemistry department and this enabled determination of functional groups of the P. harmala extract within a wavenumber range of 3600 –400 cm− 1.

Zinc oxide nanoparticle synthesis

50 mL zinc sulfate heptahydrate (ZnSO4 · 7H2O, ACS reagent, 99%, Sigma-Aldrich, USA) and 5 mL seed extract were admixed, and then agitated for 10 min in the ambient temperature, a process which produced a pale yellow solution of ZnO NPs. The admixture was then rinsed assiduously, and the resulting solution of NPs was first centrifuged (4,500 rpm, 15 min) and then dried for between 7 and 8 h at a temperature of 80 °C. When required for use, a suspension of the rough pellets in sterile double-distilled water was made up. This was then microfiltered via a 0.2 μm filter, and then placed in dark storage at 4 °C. Following dispersal in sterile double-distilled water and centrifugation, a process repeated thrice, the suspension was filtered once more.

Zinc oxide nanoparticle characterization

The X-ray diffraction (XRD) spectral features of the ZnO NPs were assessed at a wavelength of 0.154 nm, using an X-ray diffractometer (XRD-6000, Shimadzu), which encompassed a copper (Cu) K-α source of radiation, and a specimen holder which measured 20 × 0.5 mm. Particle dimensions were quantified with the use of a Malvern Zetasizer Nano ZS90. Following dilution of the stock suspensions with distilled water, the samples were allowed to equilibrate for 5 min at a temperature of 25 °C in order to ensure a uniform temperature throughout. The solution’s viscosity was 0.8872 cP, and the refractive index, 1.59.

The reduction of the zinc sulfate salt solution, and the consequent manufacture of the ZnO NPs in conjunction with the seed extract from P. harmala, were monitored using ultra-violet visible (UV-vis) spectroscopy (UV-1900, shimadzu, Japan), with double-distilled water utilized as the control. A quartz cuvette, with a 1.0 cm path distance, was used for each spectrophotometric evaluation. The wavelength spectrum, 800 –200 nm, was utilized to obtain the NPs’ surface plasmon resonance (SPR).

Scanning electron microscopy was used to study the dimensions and morphology of the ZnO NPs in more detail. After covering the sample surface with a 4 nm coat of gold, the samples were appraised in the following conditions: 50 Pa vacuum; 3 KV; 8–10 mm working distance. Furthermore, Energy-dispersive x-ray spectrometer (EDX) operated at an acceleration voltage of 20 kV was employed to analyze the elemental composition of the synthesized ZnO NPs. The above investigations were all carried out at Jordan’s University of Science and Technology in the Nanotechnology Institute.

Bacterial isolates and their identification

For this study, six clinical isolates (Pa 1 − Pa 6) of P. aeruginosa and the reference strain, ATCC 27,853, were used [26]. The clinical isolates were collected from the sputum of patients with CF attending the Prince Hamza Hospital in Jordan. In order to determine that the recovered bacteria were P. aeruginosa, the bacteria underwent various tests, including Gram staining and an oxidase test, as well as an assessment of their ability to grow at 42 °C. Bacteria were also evaluated for their motility and growth on selective medium-cetrimide agar, their synthesis of green pigments on nutrient agar, and their growth on MacConkey agar. Identification was confirmed using the VITEK2 computer automatic bacteria identification system (Bio Merière, Lyon, France). Earlier work has established that these strains are moderate-to-strong synthesizers of biofilms [27]. Antibiotic susceptibility was assessed using the disc diffusion method, which revealed that isolates Pa 3, Pa 5 and Pa 6 were resistant to cefepime. No P. aeruginosa strains exhibited multi-drug resistance. The full results of the antibiotic susceptibility of the utilized strains are shown in Supplementary Table 1.

Culture conditions

For each strain, subcultures were grown from single colonies on Pseudomonas Cetrimide agar plates. The specimens were grown in 5 mL Luria-Bertani medium and incubated at 37 °C in aerobic conditions. All strains were then stored in a LB medium containing 15% glycerol at -70 °C.

Minimal inhibitory concentration assays

In order to obtain the ZnO NPs’ minimum inhibitory concentration (MIC), a microtiter broth dilution technique was used. This is a well-established, simple, reproducible, low-cost and sensitive method [28]. The reference P. aeruginosa strain, ATCC 27,853, together with six strains that had been clinically isolated, were studied thrice in three different experiments. Each experiment required 100 µL bacteria (density, 5 × 105 CFU/mL) in MHB (Biolab, Hungary), to be pipetted into the wells of 96-well plates (tissue culture-treated polystyrene; Costar 3595, Corning Inc., Corning, NY). Different ZnO NPs concentrations were then added, from 6,000 µg /mL to 23.4 µg/mL, the latter obtained following serial double dilution. The loaded microplates were then cultured for 24 h (37 °C, 150 rpm). The reference P. aeruginosa strain, ATCC 27,853, formed the quality control. Inoculated broth alone was used as the negative control; this was incubated at the same temperature for 24 h. The MIC endpoint was defined as the lowest concentration of ZnO NPs where no growth within the well could be observed, and was confirmed with the use of a microtiter dilution method [29]reliant on a tetrazolium-based dye, which has been employed previously to identify the MIC and to study biofilms [30]. In this study, the metabolic indicator of the viability of the bacteria and biofilm development selected was triphenyl tetrazolium chloride (TTC). 40 µl 0.2 mg/mL TCC, made up to a solution with deionized water, was added to the wells. The plates were then incubated for 4 h at a temperature of 37 °C, and then ranked via the observation of color alteration within the wells, which was evaluated in the context of the positive and negative controls. Once this measurement had been completed, 50 µL aliquots from all wells in which no obvious P. aeruginosa growth was identified were seeded onto BHI agar plates. These were placed in the incubator at 37 °C for 24 h. The minimal bacteriostatic concentration was defined as the lowest ZnO NPs concentration at which 99.9% bacteria had been destroyed, and evaluated by comparing agar plate bacterial volumes prior to and following incubation.

Bacterial growth assays

Bacteria were inoculated into sterile, untreated 96-well flat-bottomed clear microtiter plates (BD Falcon), and their growth was monitored with respect to the different concentrations of ZnO NPs. Standardized suspensions of the various P. aeruginosa strain cultures were created using Tryptone Soy Broth (TSB). 20 µL bacteria was suspended in 250 µL aliquots TSB and concentrations of ZnO NPs ranging from 0.78 to 400 µg/mL. Tests were carried out in triplicate. Positive controls were created by loading the wells with bacteria and TSB only, i.e. without ZnO NPs, and the negative controls, or blanks, contained TSB alone.

The plates were then incubated in the dark for 24 h in an aerobic environment at a temperature of 37 °C. The plates were shaken for the duration. After incubation, total cell numbers were determined for each ZnO NPs concentration. Bacterial quantification was carried out by measuring optical density at 600 nm (OD600, Infinite® 200 PRO NanoQuant, TECAN). The final bacterial cell growth value for each ZnO NPs concentration was calculated, and equaled the values from the wells containing P. aeruginosa minus the average value of the blanks. The total cell growth included all cells, i.e. both biofilm-associated and planktonic.

Colony count method

Colony count method was carried out to determine the impact of ZnO NPs antibacterial activity upon P. aeruginosa strains. Similar to bacterial growth assays and after P. aeruginosa strains were incubated with ZnO NPs for 24 h, 10 µL of sample from each well was diluted by a factor of 10 until 10− 6 serial dilution was reached and plated on cetrimide agar. The experiment was conducted in a sterile PBS and cetrimide agar plates were used. Incubation of the plates were conducted for 24 h and counting was performed on the colonies to determine the bacterial viability, which is measured in CFU/mL. Equation (31)was used to calculate the CFU concentration.

$$\eqalign{{\rm{CFU}}/{\rm{mL}} & = [(\# {\rm{ of colonies }}) \times (1/{\rm{ dilution factor }}) & \cr \times 1000\mu {\rm{L}}/{\rm{mL}}]/{\rm{ (volume plated) }} \cr}$$

All the tests were conducted in triplicate and three times independently.

Bacterial metabolic activity assessment

A TTC assay was conducted in order to determine the metabolic activity of P. aeruginosa. TTC is reduced by metabolically active P. aeruginosa cells to produce a red-colored formazan derivative. This reaction enabled the colorimetric quantification of bacterial metabolic activity; absorbance was measured at 540 nm (OD540) (Knezevic and Petrovic, 2008). 50 µL 0.1% TTC (Sigma, USA) was added to the specimens to achieve a final concentration of 0.02%. Specimens were incubated at 37 °C for 2 − 4 h, and then the OD540 was measured using a VERSA max microplate reader (Molecular Devices, USA).

Quantitative determination of biofilm formation

A spectrophotometric method was used to quantify the biofilm’s total biomass, which encompassed bacterial cells and extracellular polymeric substances. 96-well flat-bottomed clear microtiter plates (BD Falcon) were used. The process followed was similar to that used for the growth assay, but the plates were not shaken during incubation. After incubating the plates for 24 h, the wells were aspirated and were washed three times in 250 µL sterile physiological saline. In order to remove loose bacteria that had not adhered to the well, the plates were then shaken vigorously.

Bacteria that remained in the plate were fixed by adding 200 µL 99% methanol to each of the wells. Plates were left for 15 min, then emptied and allowed to dry. Gram staining was performed by adding 200 µL 2% Hucker crystal violet to each well. The plates were then allowed to stand for 5 min. Excess dye was removed, and then the wells were washed thrice with 200 µL sterile water. Care was taken to not disturb the biofilm during the washing process. The plates were left to dry. Subsequently, 160 µL 33% (v/v) glacial acetic acid was applied to each well so as to solubilize the cell-bound dye.

An automated reader (ICN Flow Titertek Multiscan Plus) was used to establish the optical density (OD) of each well. Three sets of readings were taken: (i) before incubation (OD 600 nm); (ii) after incubation (OD 600 nm); (iii) and after creating the biofilm (OD 570 nm). In order to normalize the measure of biofilm formed against the growth of bacteria, a ratio of 570/600 was used. Negative OD values were presented as zero. All the experiments were conducted using three sets of samples, and each was repeated three times.

Production of extracellular polymeric substance (EPS) in the presence of ZnO NPs

Extracellular polymeric substances (EPS) are recognized to be the basic components that establish the biofilm’s physicochemical properties and consider as a key element in understanding biofilm phenotype [32, 33]. In this experiment, each P. aeruginosa strain ( 0.5 McFarland bacterial suspension) mixed with 100 ml of LB broth with ZnO NPs concentration from 7.5 to 0.225 ug/ml. The mix was incubated for 24 h at 37 °C. Preparation for control was done with no added ZnO NPs.

After incubation, EPS extraction was done from the bacterial cultures through centrifuging at 10,000 rpm at 4 °C for 30 min. To promote EPS precipitation, 1 volume of ethanol measuring at 95% was added to a batch of 3 volumes of the supernatant that was obtained from the centrifuge. This mixture is placed in the fridge for 18 h at the temperature of 4 °C.

The precipitated EPS was obtained and underwent suspension through the use of double-distilled water. All traces of bacterial cells were taken out from the suspension by filtering with cellulose acetate membrane measuring at 0.45 μm. To verify that the bacteria is indeed completely removed, a sample was placed on an agar plate and subjected to optimal condition for the growth of culture. The plate was subsequently checked for any signs of growth. The EPS that was extracted was subsequently weighed and lyophilized to determine the EPS measuring in mg EPS per 100 ml. EPS extraction experiment was performed in triplicate.

Gene expression of quorum sensing regulatory genes using a quantitative real-time polymerase chain reaction

The relative gene expression of QS-regulatory genes was estimated by using a quantitative real-time polymerase chain reaction (qRT-PCR). The total RNA from the P. aeruginosa was extracted at the middle of the log phase, corresponding to an OD600 of 0.5–0.6. RNA was recovered from the bacteria grown in MH medium at 28 °C, agitated at 50–60 rpm, in 6-well plates (Corning Inc., Corning, NY) exposed to 12.5 µg/mL ZnO NPs. Controls were cultured in the same manner, but did not include ZnO NPs. After culturing for 24–48 h, the biofilm was carefully harvested. Unbound cells were removed by gently washing the film in 10 mM sodium chloride solution. A RNeasy Mini Kit (Qiagen, Hilden, Germany) was used to extract RNA from the biofilm. Once extracted, the RNA was quantified using a Nanodrop ND-1000 instrument (Nanodrop Products Inc., Wilmington, NE). The purity of the RNA was assessed by measuring its absorbance at 260/280 nm.

A RT-PCR was performed using a BIO-RAD thermal cycler in order to synthesize cDNA at a temperature of 42 °C. Random primers, RNaseOUT, dNTPs and superscript II reverse transcriptase (Tsingke, Beijing, China) were added. A final reaction volume of 20 µL was created by adding 2 µL template DNA to 0.5 µL of both forward and reverse primers, 10 µL Luna Universal qPCR Master Mix and 7 µL nuclease-free water.

The following protocol was adopted: pre-denaturation, at 95 °C for 3 min; denaturation, 34 cycles, at 94 °C for 30 s; annealing, at 50 –63 °C for 30 s; elongation, at 72 °C for 1 min; final extension, at 72 °C for 5 min. Agarose gel electrophoresis was used to verify PCR amplification, and the relative expression of QS-regulating genes was evaluated against the housekeeping gene, rpoD. The relative level of gene expression in the cultures treated with ZnO NPs was compared against the untreated cultures using the 2-∆∆Ct method. Details of the primer sequences are presented in Supplementary Table 2.

Ethical approval

Ethical approval for this study was given by the ethics service committees from Hashemite University and Prince Hamza Hospital (Ref No. 710/2019–2020). The experiments were carried out in keeping with pertinent legislation. All patients with CF from whom clinical isolates of P. aeruginosa were obtained gave informed consent.

Statistical analysis

Results of the experiments are presented as the mean ± standard error of mean (SEM) of the three replicates. One-way analysis of variance (ANOVA) was used to analyze the differences between controls and the cultured samples. Tukey’s test was used for the comparison of the OD values derived from the microtiter-plate tests for the different concentrations of ZnO NPs, as well as the control samples,. The threshold for statistical significance was set as P < 0.05. The data were analyzed using GraphPad Instat 6.0 software.

Results

Fourier transform infrared spectroscopy (FT-IR) analysis

Fourier Transform Infrared Spectroscopy (FT-IR) was used to determine organic and inorganic compounds of the seeds extract of the P harmala. The general purpose of FTIR analysis is to determine the presence of vibration of function groups within the organic molecules. Therefore, the resultant peaks determine the presence of functional groups within the organic and inorganic compounds. The extraction of P.hermala seed was subjected to FTIR spectroscopic analysis to determine the existence of various chemical constituents (Fig. 1). The FTIR findings showed four major characteristic peaks for P. harmala seed extract.

Fig. 1
figure 1

Fourier transform infrared spectroscopy (FTIR) spectra of the P. harmala seeds extract

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The first peak for the P. harmala extract spectrum was at 3284.32 cm− 1, and this was delegated to the OH-stretching of hydroxyl groups such as phenols, alcohols and NH group of amides or amines. The second peak for the P. harmala extract spectrum was at 1612.55 cm− 1, and this was delegated to the C = O group of carboxylic acids. The peak of strong absorption was at 1574.77 cm− 1 occur as a result N-H bending vibration. The final peak for P. harmala extract spectrum at 1025.46 cm− 1 and was delegated to C = CH2.

Zinc oxide nanoparticle biosynthesis

The hue of the solution containing zinc sulphate and the seed extract of P. harmala transformed from a light brown to a yellowish black during the formation of the ZnO NPs, reflecting the reduction of zinc ions as the ZnO NPs were generated.

UV-vis spectrophotometry, with a wavelength spectrum of between 800 nm and 200 nm, was utilized to quantify the ZnO-NPs’ SPR, which is evident in the range, 350–450 nm, with a prominent absorption zenith at 399 nm (Fig. 2A).

Fig. 2
figure 2

Characterization of ZnO NPs: (A) ultraviolet visible spectroscopy data; (B) scanning electron microscopy (SEM) image (x 500 magnification) showing morphology (yellow circles indicate ZnO NPs’ circumference); (C) ZnO-NPs particle size distribution using SEM data; (D) X-ray diffraction data for ZnO-NPs; (E) dynamic light scattering spectrum showing ZnO-NPs particle size distribution; (F) ZnO-NPs zeta potential analysis; (G) The EDX results of ZnO- NPs

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The SEM imags of the ZnO NPs revealed that the particles were consistently formed and sized, with an average measurement of 50 nm. This is seen in Fig. 2B. Figure 2C shows the size distribution of ZnO-NPs. The histograms show that the ZnO-NPs created in this research have a predominant particle size of 49.8 ± 12.6 nm.

Figure 2D displays a hexagonal wurtzite ZnO structure with crystal planes corresponding to the Miller indices (h, k, l): (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), (202) in the pure ZnO-NPs sample. The dimensions were determined to be 44 nm by analyzing the prominent diffraction peaks, namely 101, using the Debye-Scherrers equation. This implies that the ZnO-NPs crystals have a high level of purity and do not include any other contaminants in their crystal structure.

The mean particle size of ZnO-NPs was determined to be 48 nm using DLS (Fig. 2E). Zeta potential is the electrical potential that occurs due to the movement of nanoparticles and solvent at the boundary between solid and liquid. Electrical potential and surface charge affect the stability of nanoparticles. The ZnO-NPs had a zeta potential of -34.7 mV as shown in Fig. 2F. EDX spectrum (Fig. 2G) show peaks for only zinc and oxygen which demonstrated that no other impurity is preset in the sample, and hence confirm the formation of ZnO.

Minimum inhibitory concentration and minimal bacteriostatic concentrations MIC

The turbidity, indicative of bacterial growth of the P. aeruginosa strains, of the test tubes containing between 23.4 µg/mL and 1.500 µg/mL ZnO NPs was inspected following 24 h incubation at a temperature of 37 °C in aerobic conditions. No turbidity, representing absent bacterial growth, was seen in test tubes containing concentrations of ZnO NPs between 3000 µg/mL and 6000 µg/mL. The suspensions from the latter tubes underwent inoculation on to a BHI agar plate, and incubation for a period of 24 h. The plate with the 6000 µg/mL ZnO NPs concentration had no bacterial growth, demonstrating a bactericidal effect of this ZnO NPs strength. It can be concluded that at the respective ZnO NPs concentrations of 3000 µg/mL and 6000 µg/mL, the MIC and MBC for all the investigated strains of P. aeruginosa were achieved.

Impact of zinc oxide nanoparticles on the growth of P. aeruginosa

The impact of ZnO NPs, using concentrations ranging between 0.78 µg/mL and 400 µg/mL, against P. aeruginosa strains, i.e. ATCC 27,853, and the six clinical isolates, that exhibited resistance to various antibiotics was evaluated. The growth of ATCC 27,853, Pa 1, Pa 2, Pa 3, Pa 4 and Pa 5 was significantly inhibited following incubation with 400 µg/mL ZnO NPs concentration (F [6, 77] = 86.68, P < 0.001); between 8.6% and 18.5% of the bacteria were inhibited. In contrast, the growth of Pa 6 was not inhibited by this concentration of ZnO NPs. Furthermore, Pa 2 and Pa 4 demonstrated 14.3% and 11.3% inhibition, respectively, following exposure to a ZnO NPs concentration of 200 µg/mL (F [10, 11] = 20.17, P < 0.001). The general trend across all strains was reduced growth at ZnO NPs concentrations > 100 µg/mL(Fig. 3).

Fig. 3
figure 3

Impact of ZnO NPs concentrations, ranging from 0.78 µg/mL to 400 µg/mL (x-axis), on the growth of the ATCC 27,853 strain and the six clinically isolated strains (Pa 1- Pa 6) of P. aeruginosa, quantified by measuring the absorbance at 600 nm (y-axis), following 24 h of incubation. *statistically significant inhibition, (***P < 0.0001, **P < 0.001, *P < 0.01), # statistically significant stimulation (##P < 0.001, #P < 0.01)

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On the other hand, Bacterial growth increased significantly for some strains when the dose of ZnO NPs was low. ATCC 27,853, Pa 3 and Pa 4 grew, i.e. 13.5%, 6.8% and 8.4%, respectively, when the concentration of ZnO NPs was 25 µg/mL (F [1, 18] = 25.04, p < 0.001). At 12.5 µg/mL and 6.25 µg/mL ZnO NPs concentrations, the growth of ATCC 27,853, Pa 2, Pa 4 and Pa 5 was stimulated at a statistically significant level. Pa 3 growth was increased significantly at concentration 12.5 µg/mL. These findings were statistically significant (F [10, 11] = 15.6, P < 0.001) (Fig. 3). The percentage data for growth inhibition are presented in Supplementary Table 3. The growth of all strains was comparable to controls when the concentration of ZnO NP was < 6.25 µg/mL. There was a general decline in growth when the ZnO NPs concentration was > 100 µg/mL, and an increase in growth when the concentration of NPs was between 3.12 µg/mL and 25 µg/mL.

Bacterial colony count method

The viable concentrations of bacterial cells were determined by enumerating colony-forming units (CFUs) before and after exposure to the ZnO NP. Similar to the results of the turbidity method, the results of the bacterial colony counting demonstrated a significant (F [10, 11] = 10.42,p < 0:05) dose-dependent reduction in the number of bacterial colonies for all the tested strains apart o f PA5 and PA6 at concentration of 400 and 200 µg/mL, PA5 only affected at statistically significant level at concentration of 400 µg/mL. of ZnO NPs at 24 h as compared to negative control (Fig. 4).

Fig. 4
figure 4

Impact of ZnO NPs concentrations, ranging from 0.78 µg/mL to 400 µg/mL (x-axis), on the viable concentrations of the ATCC 27,853 strain and the six clinically isolated strains (Pa 1- Pa 6) of P. aeruginosa, quantified by enumerating colony-forming units (CFUs) before and after exposure to the ZnO NP (y-axis), following 24 h of incubation. *statistically significant inhibition, (***P < 0.0001, **P < 0.001, *P < 0.01), # statistically significant stimulation (##P < 0.001, #P < 0.01)

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However, when the concentration ranged at 6.25–25 µg/mL, there is a statistically increase in the number of bacterial colonies being observed (F [10, 11] = 19.78, P < 0.0001)(Fig. 4). When ZnO NPs concentration was at 12.5 µg/mL and 25 µg/mL there is a significant increase of ATCC strain and PA3 strain colony respectively. There is a significant increase for PA1, PA2 and PA5 colony when concentration was between 6.25 µg/mL and 12.5 µg/mL. P4 colony increase significantly when concentration was between 6.25 µg/Ml to 25 µg/mL. Example of the impact of ZnO NPs on ATTCC strai is provided in supplementary Fig. 1.

Impact of zinc oxide nanoparticles on the metabolic activity of P. aeruginosa

An analysis of the metabolic activity of planktonic P. aeruginosa is presented in Fig. 5. The metabolism of planktonic ATCC 27,853, Pa 1, Pa 2, Pa 4, Pa 5 and Pa 6, was significantly inhibited by the highest concentration of ZnO NPs, 400 µg/mL (F [10, 11] = 10.94, P = 0.0002). Pa 1 and Pa 5 were also affected when the concentration of ZnO NPs was ≥ 200 µg/mL.

Fig. 5
figure 5

Impact of ZnO NPs on the metabolic activity of P. aeruginosa planktonic cells. The latter is denoted by absorbance at 600 nm (y-axis) 4 h after TCC stain application. The six clinically isolated strains (Pa 1-Pa 6) are illustrated at ZnO NPs concentrations varying between 0.78 µg/mL and 400 µg/mL (x-axis) following 24 h of incubation. *statistically significant inhibition, (***P < 0.0001, **P < 0.001, *P < 0.01), # statistically significant stimulation (###P < 0.0001, (##P < 0.001, #P < 0.01)

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Compared to controls, bacterial metabolic activity increased in the presence of moderate concentrations of ZnO NPs. ATCC 27,853 and Pa 1 were stimulated at 6.25 µg/mL, whereas Pa 2 and Pa 4, and Pa 5 and Pa 6, were affected by concentrations of ZnO NPs ranging from 6.25 µg/mL to 25 µg/mL, and 6.25 µg/mL and 12.5 µg/mL, respectively. The metabolic activity of Pa 3 was stimulated compared to control. however, this was not at statistically significant level.

Impact of zinc oxide nanoparticles on biofilm formation

As shown in Fig. 6, biofilm formation of all P. aeruginosa strains was inhibited when the concentration of ZnO NPs was 400 µg/mL (F [10, 11] = 13.04, P < 0.001) with a general decline of the biofilm biomass when the concentration of NPs was > 50 µg/mL. Interestingly, when the concentration of ZnO NPs was 12.5 µg/mL, the biofilm biomass of ATCC 27,853 and Pa 1 strains increased (F [10, 11] = 8.379, P = 0.008). The biofilm biomass of Pa 4 increased significantly following exposure to a ZnO NPs concentration of 6.25 µg/mL (F [10, 11] = 7.824, P = 0.0011). A biphasic response was observed in all strains except for Pa 6. The percentage of growth inhibition is presented in Supplementary Table 4.

Fig. 6
figure 6

Impact of ZnO NPs concentrations, ranging from 0.78 µg/mL to 400 µg/mL, on biofilm formation for ATCC 27853 and the six clinically isolated strains (Pa 1-Pa 6) after a 24-hour incubation period. *statistically significant inhibition, **P < 0.001, *P < 0.01), # statistically significant stimulation (##P < 0.001, #P < 0.01)

Full size image

Effect of ZnO NPs on EPS production

P. aeruginosa strains were cultured with and without the presence or with subinhibitory concentrations of ZnO NPs to determine its impact on the production of EPS. Figure 7 shows the EPS extracted weight from all strains. The weight is significantly lower in comparison to the culture grown without ZnO NP concentration at 400 µg/mL among all strains (F [10, 22] = 10.45, P < 0.0001). However, there is a significant stimulated production of EPS for ATCC, PA1, PA3, PA4 and PA6 strains when ZnO NPs concentration was at 6.25 and 12 µg/mL. 25 µg/mL concentration of ZnO NPs also stimulate production of EPS among ATTCC strain significantly.

Fig. 7
figure 7

Impact of ZnO NPs concentrations, ranging from 0.78 µg/mL to 400 µg/mL, on EPS production for ATCC 27853 and the six clinically isolated strains (Pa 1-Pa 6) after a 24-hour incubation period. *statistically significant inhibition, **P < 0.001, *P < 0.01), # statistically significant stimulation (##P < 0.001, #P < 0.01)

Full size image

Impact of zinc oxide nanoparticles on quorum sensing-regulated gene expression

In order to evaluate the expression of the QS-regulated genes, lasI, lasR, rhlI, rhlR, pqsR, and pqsA, the calculated Ct values for the genes were used. The RopD reference gene was taken as the average standard against which gene expression was assessed. Figure 8 shows the relative expression of QS-regulated genes in the P. aeruginosa strains exposed to 12.5 µg /mL ZnO NPs compared to the ZnO NPs-free controls.

Fig. 8
figure 8

The effect of 12.5 µg/mL ZnO NPs on the relative expression of QS-regulated genes in P. aeruginosa strains compared to control samples not exposed to Zn NPs. *statistically significant stimulation (*P < 0.01)

Full size image

Compared to control samples not treated with ZnO NPs, the expression of LasB in ATCC 27,853, Pa 1 and Pa 4 treated with ZnO NPs was increased significantly (F [1, 28] = 41.46, P < 0.001). Both LasI and RhII upregulation were identified in treated samples of ATCC 27,853 and Pa 4. Expression of LasI and RhII gene were also increase in Pa 3 and Pa 2, respectively. ATCC 27,853, Pa 1 and Pa 6 strains demonstrated a statistically significant higher LasR expression when incubated with ZnO NPs.

Discussion

P. aeruginosa is well-established as a major cause of hospital-acquired infections, and so there is a pressing need to implement efficacious preventative infection control strategies [34]. The bacterium has a range of mechanisms which enable it to exhibit both natural and acquired resistance to a number of antibiotics [35]. Over the last ten years, studies on the anti-microbial and catalytic characteristics of NPs have demonstrated their superlative activity in this regard, and have piqued notable interest in their potential clinical applications [36]. ZnO NPs are already widely used in the commercial sector [37]. The current study has focused on the effectiveness of ZnO NPs as anti-microbial agents with respect to clinical isolates of P. aeruginosa.

Seed extract from P. harmala was used to biosynthesize the ZnO particles utilized in this study. Green synthesis methods exploit reducing and capping compounds, which facilitate nanoparticle stability, obtained from natural plant extracts. Plant extracts behave as reducing and capping agents; the latter enhance the stability of NPs and influence their morphology. Examples of substances used include phenolics, polysaccharides, flavones, terpenoids, alkaloids, proteins, amino acids, enzymes and alcoholic compounds obtained from plants [38, 39]. Their utilization reduces the need for reagents that are both costly and potentially toxic [40], and limits the amount of waste and environmental contamination that arises when chemically derived compounds and solvents are used [41]. Consequently, biosynthesis is a cost-effective and straightforward technique, which does not need any complex apparatus, and yields NPs that are biocompatible and have a minimal effect on the environment [40].

It was determined that during the green synthesis process, exposure of zinc sulfate to the seed extract obtained from P. harmala led to the formation of ZnO NPs, as indicated by the change in solution color and the UV-vis spectroscopy data. ZnO-NPs are direct band-gap semiconductors with a wide band-gap of approximately 3.37 eV between the conduction and valence bands [42]. This results in an absorption peak between 350 and 450 nm for ZnO-NPs when electrons jump from the valence band to the conduction band [43]. The UV-Vis spectra displays a significant absorption peak at around 399 nm for ZnO-NPs as seen in Fig. 1. On SEM, the morphology of the ZnO NPs were observed to be relatively consistent, and approximately spherical; mean diameter was 50 nm. Previous studies have demonstrated a negative association between the anti-microbial effects of ZnO NPs and their dimension, with a less potent effect observed the greater the nanoparticle diameter above 100 nm [44]. Bactericidal properties were observed in ZnO NPs with the smallest diameter, whereas larger entities exhibited bacteriostatic effects [45].

EDX spectrum show peaks for only zinc and oxygen which demonstrated that no other impurity is preset in the sample. This validates that ZnO was formed. Such findings are in accordance with other studies that conducted various experiments in synthesizing ZnO in various forms. When comparing the existing results with the results from literature, the conclusion is that the effective synthesis of ZnO NPs is through this study’s method. Reviewing the data from the literature against the current results, it was concluded that ZnO NPs were effectively generated by the method used in this study.

Earlier research has shown that the growth and formation of P. aeruginosa biofilms is impaired when the bacteria are challenged by ZnO NPs, and so it can be concluded that ZnO NPs exhibit anti-bacterial activity [46,47,48]. Earlier work from this group demonstrated that ZnO NPs, biosynthesized from olive leaves, were effective against the growth and biofilm formation of P. aeruginosa [27]. Building on the previous study, the current findings indicate that not only do relatively high concentrations (100–400 µg/L) of ZnO NPs possess anti-bacterial qualities, they also have anti-biofilm effects in relation to P. aeruginosa. The growth, biofilm formation and metabolic activity of some strains of P. aeruginosa were diminished when challenged by ZnO NPs at concentrations of 100–400 µg/mL. However, bacterial growth increased when the concentration of ZnO NPs was between 6.25 µg/L and 25 µg/L. Similarly, high concentrations of ZnO NPs exerted inhibitory effects on biofilm biomass and metabolic activity, but low concentrations induced a higher biomass and increased metabolic activity.

Hormesis, which is considered to be an adaptive function, describes a biphasic dose-response relationship, whereby low concentrations of an agent stimulate a response, which is inhibited at high concentrations of the same agent [49, 50], as observed in this study, with respect to the ZnO NPs. Following exposure to diverse stimuli, dose-dependent biphasic responses have been observed in multiple biological systems, and in different organisms [51, 52]. It has been reported that plants respond to antibiotics in a hormetic manner [53]. Although some studies describe that antibiotics may stimulate a hormetic response in Gram-negative bacteria [54], there do not appear to be any studies that have investigated metal NPs-induced hormetic responses relating to Gram-negative bacteria, and specifically, to P. aeruginosa. Studies of hormetic responses in bacteria exposed to antibiotics suggest that such responses comprise specialized survival mechanisms, and facilitate exponential growth at low concentrations of anti-microbial agents. Studies exploring the way in which NPs may initiate hormetic responses have been conducted [55, 56]. However, the mechanisms through which NPs can achieve such a response in bacterial cells have yet to be elucidated.

Similar to the findings of the current work, previous studies, which have focused on different NPs and bacteria, have highlighted a biphasic hormitic response to NPs [57,58,59,60]. For example, Schacht, Neumann [58] observed that the growth of Cupriavidus necator H16 test organisms was stimulated by silver (Ag) NPs (< 15 nm) at concentrations of 20–60 µg/mL. Xiu, Zhang [59] found that at a concentration of 1.8–2.2 µg/mL, Ag NPs promoted the growth of E. coli, by 6−13%, rising to 11–21% with Ag NPs concentrations of 5.7–16.4 µg/mL. Furthermore, the ability of V. cholerae to form biofilms was enhanced in the presence of ZnO NPs measuring between 90 and 100 nm [57]. E. coli growth and biofilm formation also increased in the presence of single-walled carbon nanotubes at concentrations of 5–300 µg/mL [61]. In the present study, P. aeruginosa growth was augmented when the sublethal concentration of ZnO NPs was in the range, 6.25–25 µg/mL. These low-dose concentrations were 0.20–0.83% of the individual MIC values for the respective bacterial strains.

Interestingly, in this research it was observed that when the concentration of ZnO NPs was 12.5 µg/mL, the biofilm biomass of ATCC 27,853 and Pa 1 strains increased. The biofilm biomass of Pa 4 became elevated significantly at a ZnO NPs concentration of 6.25 µg/mL. It is possible that enhanced biofilm growth may occur as a defense strategy when bacteria are exposed to more dilute ZnO NPs strengths, thereby offering protection from anti-microbial compounds [62]. In order to examining the effect of ZnO NPs on QS, qRT-PCR was used to study the relative expression of QS regulatory genes which govern P. aeruginosa biofilm formation. In earlier work from this group, application of 900 µg/mL ZnO NPs downregulated lasI, lasR, rhlI, rhlR, pqsR, and pqsA gene expression. In clinical isolates of P. aeruginosa, Abdelraheem and Mohamed [63] found that both biofilm and virulence genes, i.e. LasR, rhII, pqsR, lecA, pela, exoS and lasA, demonstrated significant expression downregulation in response to ZnO NPs. However, the results from the current study suggested that more dilute ZnO NPs concentrations had a stimulatory effect on several QS governing genes; these data are contrary to those documented at higher ZnO NPs solution strengths. Previous studies have also observed a stimulatory effect on biofilms and QS genes for several antimicrobial agent including NPs. For instance, exposure of Staphylococcus epidermidis to tetracycline in a subinhibitory concentration induced polysaccharide intercellular adhesin gene expression amplification, and biofilm generation was promoted by other stressors, including osmolarity and heat [64, 65]. In Escherichia coli [66]and P. aeruginosa [67], the growth of biofilms was enhanced by nickel ions in solution, and mucin, respectively. The overall process of QS inhibition or stimulation by NPs, and the mechanisms occurring at a molecular level, require further elucidation. The current study has identified a serendipitous finding linked with the application of sub-bactericidal strengths of ZnO NPs, i.e. QS gene expression amplification and the consequent promotion of biofilm growth. Given that the elimination of biofilms is more challenging than that of planktonic cells [68], the latter finding implies that NPs could augment both the virulence and disease-inducing capacity of bacteria, and highlights the requisite for assiduous ZnO NPs usage, and governance of their implementation within the clinical arena.

Drawing on the evidence reported in the literature and the results of the current study, it can be speculated that low concentrations of ZnO NPs could stimulate bacterial production of sublethal levels of reactive oxygen species (ROS), which could, in turn, induce bacterial adaptation and cellular proliferation. Studies have suggested that sublethal levels of ROS cause the expression of ROS scavenger enzymes and the upregulation of antioxidant mechanisms; these adaptive responses to oxidative stress support bacterial survival [69, 70]. In order to protect itself against ROS, P. aeruginosa synthesizes two superoxide dismutases (SODs) reduce O2− to H2O2 and O2. P. aeruginosa also safeguards itself with three catalases, i.e. KatA, KatB and KatC [6,32], and four alkyl hydroperoxide reductases, i.e. AhpA, AhpB, AhpCF and Ohr [71,72,73,74]. These systems may facilitate the survival of P. aeruginosa at low concentrations of ZnO NPs. There is a close relationship between biofilm formation and oxidative stress, which arises from the fact that the same upstream metabolic pathway regulators are involved in both processes [75]. ROS act on the metabolic pathways to modulate the physiology of the bacterial biofilm by altering its characteristics, morphology and structure (76). In this study, low concentrations of ZnO NPs promoted biofilm biomass and metabolic activity.

Although the precise mechanisms that facilitate the enhanced growth and biofilm formation of P. aeruginosa remain unknown at this juncture, it is possible that the bactericidal effects of the ZnO NPs arise following dissolution of their crystalline core, which releases soluble zinc ions (Zn2+) into the medium [77]. Inside bacterial cells, Zn+ 2 ions interact with the negatively charged phosphate backbone of the DNA, which damages the DNA and stimulates excessive ROS production [78, 79]. The stress initiated by the high concentration of Zn+ 2 ions overwhelms bacterial defense mechanisms, thereby failing to eliminate the ROS. Ultimately, redox homeostasis is lost as ROS production becomes uncontrolled. Without redox homeostasis, the cell membrane undergoes lipid peroxidation, and replication, transcription and translation mechanisms are damaged, trauma that leads to cell death [17, 80]. However, when Zn+ 2 ion concentrations are low, the bacterial defense mechanisms are not overwhelmed, and so are able to respond to the toxic threat presented by the ZnO NPs. The genotoxic damage caused by the ZnO NPs gives rise to an abnormal massing of ssDNA in the cell, which triggers a ‘save our souls’ response; this, in turn, stimulates DNA repair mechanisms [81, 82]. The repaired DNA gives rise to DNA repair heterogeneity, enabling the cell to survive. This latter is a stress-acquired feature that arises either from variations in the genes that regulate proteins, such as DNA-dependent kinase, or the accumulation of mutations and rearrangement of the genome [83]. Persister cells have the ability to withstand the toxicity of the sub-lethal dose of ZnO NPs, and they are potentiated in order to survive. Stimulation of their surge overcompensates for cell damage and population size [84]. The homeostatic system that initiates compensatory mechanisms to restore the system to the set point demands the coordination of multiple cellular processes. These processes operate synergistically, giving rise to greater numbers of persister cells with superior stress-resistance capabilities [85, 86]. This explanation, which is drawn from data presented in recently published literature, might be a plausible account of the hormetic response described in this study. Further research is warranted to verify the current findings and to determine the biochemical or molecular pathways involved in promoting cell growth.

In conclusion, this study supports the results of earlier work and confirms that specific concentrations of ZnO NPs have anti-bacterial properties, thereby inhibiting bacterial growth and biofilm formation. However, a hormetic effect is observed at low concentrations of ZnO NPs. Although the molecular mechanisms underpinning hormesis have yet to be fully elucidated, it is recognized that hormesis is a generalized adaptive response to nanoscale xenobiotic challenges. Further research is warranted to characterize the risks of using low-dose NPs to treat different types of bacteria. Additionally, since P. aeruginosa is ubiquitous microorganism and present in every environment, this research adds to the understanding of the interaction of this bacteria underlying the effect of exposing this bacterium to metal NPs at low dose at the environment. Therefore, the impact of using low NPs concentrations in both natural and artificial environments or systems should be pursued with caution.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

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Acknowledgements

The authors would like to thank the administrative and healthcare staff of The Prince Hamza Hospital for their cooperative efforts throughout the conduction of this study.

Funding

H Al-Momani received financial support from the Department of scintifc reaserch at The Hashemite University/Jordan.

Author information

Authors and Affiliations

  1. Department of Microbiology, Pathology and Forensic Medicine, Faculty of Medicine, The Hashemite University, P.O box 330127, Zarqa, 13133, Jordan

    Hafez Al-Momani

  2. Department of Anatomy, Physiology and Biochemistry, Faculty of Medicine, The Hashemite University, P.O box 330127, Zarqa, 13133, Jordan

    Iman Aolymat

  3. Faculty of Applied Medical Sciences, The Hashemite University, Zarqa, 13133, Jordan

    Lujain Ibrahim, Hadeel Albalawi, Dua’a Al Balawi & Muna Almasri

  4. Nanotechnology Institute, Jordan University of Science & Technology, P.O. Box 3030, Irbid, 22110, Jordan

    Borhan Aldeen Albiss

  5. Medical Laboratory Department, Prince Hashem Military Hospital, Zarqa, 13133, Jordan

    Sahar Alghweiri

Authors
  1. Hafez Al-Momani
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  2. Iman Aolymat
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Contributions

H. Al-Momani was responsible for the study design, formal analysis, investigation, supervised D. A, M. A, L. I and H. A for performing the experiments. H. Al-Momani prepared figures and Writing—original draft. D. A, M. A, L. I and H. A: all are research assistance and they responsible for performing the experiments under supervision of H. Al-Momani. S Al: responsible for PA clinical strains isolation and identification. I. A and B A: responsible for biosynthesis of Zinc Oxide nanoparticles and Nanoparticles characterization. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Hafez Al-Momani.

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Ethics approval and consent to participate

Ethical approval for this study was given by the ethics service committees from Hashemite University and Prince Hamza Hospital (Ref No. 710/2019–2020). The experiments were carried out in keeping with pertinent legislation. All patients with CF from whom clinical isolates of P. aeruginosa were obtained gave informed consent.

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Not applicable.

Competing interests

The authors declare no competing interests.

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Al-Momani, H., Aolymat, I., Ibrahim, L. et al. Low-dose zinc oxide nanoparticles trigger the growth and biofilm formation of Pseudomonas aeruginosa: a hormetic response. BMC Microbiol 24, 290 (2024). https://doi.org/10.1186/s12866-024-03441-y

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Keywords

BMC Microbiology

ISSN: 1471-2180

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