Ó 2014 Eur J Oral Sci

Eur J Oral Sci 2014; 1–7 DOI: 10.1111/eos.12152 Printed in Singapore. All rights reserved

European Journal of Oral Sciences

Anti-biofilm and antibacterial activities of zinc oxide nanoparticles against the oral opportunistic pathogens Rothia dentocariosa and Rothia mucilaginosa

Shams T. Khan1, Maqusood Ahamed2, Javed Musarrat3, Abdulaziz A. Al-Khedhairy1 1

Department of Zoology, College of Science, King Saud University, Riyadh; 2King Abdullah Institute for Nanotechnology, King Saud University, Riyadh, Saudi Arabia; 3 Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India

Khan ST, Ahamed M, Musarrat J, Al-Khedhairy AA. Anti-biofilm and antibacterial activities of zinc oxide nanoparticles against the oral opportunistic pathogens Rothia dentocariosa and Rothia mucilaginosa. Eur J Oral Sci 2014; 00: 000–000. © 2014 Eur J Oral Sci Species of the genus Rothia that inhabit the oral cavity have recently been implicated in a number of diseases. To minimize their role in oral infections, it is imperative to reduce and/or control the growth and biofilm formation activity of Rothia spp. In this study, two bacterial isolates, Ora-7 and Ora-16, were obtained from the oral cavity of a healthy male subject and identified as Rothia dentocariosa and Rothia mucilaginosa, respectively, using a polyphasic taxonomic approach. Antimicrobial and anti-biofilm formation activities of zinc oxide nanoparticles (ZnO-NPs), of average size 35 nm, were assessed in in vitro assays using Crystal Violet and live and dead staining techniques. The ZnO-NPs exhibited an IC50 value of 53 and 76 lg ml1 against R. dentocariosa and R. mucilaginosa, respectively. Biofilmformation assays, performed on the surfaces of polystyrene plates, artificial teeth, and dental prostheses, revealed the efficacy of ZnO-NPs as a potential antibacterial agent for controlling the growth of Rothia isolates in both planktonic form and biofilm.

The genus Rothia consists of six species, three of which, viz. Rothia dentocariosa, Rothia mucilaginosa, and Rothia aeria, are common inhabitants of the oral cavity and are essentially benign. Recent reports suggest these species to be opportunistic pathogens, causing a number of diseases in addition to dental and periodontal ailments (1). R. dentocariosa, originally isolated from carious lesions of human teeth, has been found to cause endocarditis (2), pneumonia (3), and infections of the peritoneum and lung (4). Similarly, R. mucilaginosa isolated from mouse (5) has been reported to cause bacteraemia (6) and pneumonia (7). CHAVAN et al. (8) have shown that infection with R. mucilaginosa in children with haematological malignancies or following haematopoietic stem cell transplantation results in a higher rate of mortality, and R. aeria (isolated from the air of a Russian space laboratory) has been reported as the causative agent of acute bronchitis (9) and sepsis in neonates (10). Therefore, these early studies have suggested that Rothia spp. are infective opportunistic pathogens, especially in immunocompromised patients (3, 11, 12). Because Rothia infections pose a much more serious health issue than

Shams Tabrez Khan, Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia E-mail: [email protected] Key words: anti-biofilm activity; antimicrobial activity; Rothia sp.; zinc oxide nanoparticles Accepted for publication September 2014

previously thought, the development of simple and effective approaches to minimize and/or prevent their infectivity is crucial. Growing resistance of bacteria to traditional antibiotics is a critical problem, even amongst oral bacteria (13), which is further complicated by the formation of biofilm on oral surfaces by these bacteria owing to the fact that biofilms exhibit significantly greater resistance to antibiotics than do planktonic cells, resulting in the development of chronic and recurring infections. Recently, metal oxide nanoparticles (NPs) have been regarded as promising alternatives to traditional antimicrobial agents (14–17). A number of reports have demonstrated the antimicrobial and antibiofilm activities of NPs against pathogenic bacteria (16, 17). Zinc oxide NPs (ZnO-NPs) have been reported to exhibit remarkable antimicrobial and anti-biofilm activity against oral bacteria (14), and are known to inhibit dentine demineralization (18). In this study, we investigated the antimicrobial and anti-biofilm potential of ZnO-NPs against the oral bacterial isolates R. dentocariosa (Ora-7) and R. mucilaginosa (Ora-16).

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Material and methods Preparation of ZnO-NPs The ZnO-NPs were synthesized using the Sol-gel method and were characterized by transmission electron microscopy (TEM) (JEM-2100F; JEOL, Akishima, Tokyo, Japan) and X-ray diffraction (XRD; Spectris, Egham, Surrey, UK). Details of the synthesis and characterization of ZnO-NPs have been discussed in an earlier publication (14). Powdered ZnO-NPs (10 mg ml1) were suspended in Milli-Q water and were sonicated for 15 min at 40 W, using a sonicator (Pro Scientific, Oxford, CT, USA), to form a homogeneous suspension before the treatments.

(Bio-Rad Laboratories, Hercules, CA, USA). The amplicons were sequenced using an automated ABI-Prism 377 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The 16S rRNA sequences thus obtained were compared with the homologous sequences retrieved from GenBank using BLASTN (23). Multiple sequence analysis was performed using CLUSTALX with default parameters (24). The phylogenetic trees were constructed using the Neighbor joining (NJ) method with nucleotide pairwise genetic distances corrected with the Kimura two-parameter method (25) using MEGA6. The reliability of the tree’s topology was subjected to a bootstrap test, and numbers at nodes indicate bootstrap support values calculated from 1,000 replications. Bootstrap values of < 700 were judged as inconclusive and are not shown in the tree.

Isolation of Rothia spp. Samples from the oral cavity of a 40-yr-old man were collected using a sterile cotton swab and suspended in autoclaved phosphate-buffered saline (PBS; pH 7.0). Appropriate dilutions in PBS were spread on brain–heart infusion agar (BHI; Mast Group, Bootle, UK) supplemented with 2% sucrose. The BHI agar plates were incubated for 48 h at 37°C. Pure cultures were obtained by picking up single, smooth, white-coloured colonies and repeated streaking on Petri dishes containing fresh BHI agar. Purified isolates were maintained on Petri dishes containing BHI agar and were stored at 80°C in 20% glycerol. Biochemical characterization of Rothia isolates Pure cultures of Rothia isolates were tested for a number of key characteristics using standard procedures (19). The Gram reaction was performed using Gram staining and the potassium hydroxide (KOH) string test (20). The presence of catalase, acid production on triple sugar iron agar, gelatin hydrolysis, haemolysis, and acid production from glucose, lactose, and sucrose, were checked following the protocols described by COWAN & STEEL (21). Growth in nutrient broth in either the absence or the presence of 6% NaCl was checked. Also, growth at different temperatures (25, 35, and 42°C) was tested on BHI agar. 16S rRNA gene-based phylogenetic analysis The 16S rRNA gene was amplified and sequenced, as described by KHAN et al. (22). Genomic DNA of isolates was obtained using Prepman Ultra (Applied Biosystems, Carlsbad, CA, USA) and stored at 20°C. The genes coding for 16S rRNA were amplified employing the primer pair 8F (50 -AGAGTTTGATCCTGGCTCAG-30 )/1492R (50 -CGGTTACCTTGTTACGACTT-30 ). Briefly, the genomic DNA (~50 ng) was used as a template in a 50 ll PCR mixture containing 0.20 mM deoxyribonucleotide triphosphates (dNTPs), 25 pmol of each primer, 10 ll of 59 Taq polymerase buffer, 2.0 mM MgCl2, and 0.2 U Taq DNA polymerase. The PCR parameters for gene amplification consisted of 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min 30 s with an initial denaturation and final extension for 5 and 10 min at 94 and 72°C, respectively. Amplification was performed on a Primus 25 Advanced PCR machine (PEQLAB Biotechnologie, Erlangen, Germany). The PCR products were analysed on a 1.2% agarose gel containing ethidium bromide and documented on a Bio-Rad Universal Hood II gel documentation system

Effect of ZnO-NPs on growth of Rothia isolates Cultures of both Rothia isolates were grown overnight in BHI broth. Aliquots of 500 ll were added to 5 ml of sterile BHI broth containing different concentrations (50, 100, 200, 300 and 500 lg ml1) of ZnO-NPs. Broth lacking ZnO-NPs was used as the control. The cultures were incubated at 37°C for 16 h and appropriate dilutions of treated and untreated cells were spread on BHI agar plates. The plates were incubated at 37°C for 3 d, and the numbers of colony-forming units (CFUs) were determined and presented as log10 CFUs ml1. Live and dead staining Live and dead staining was performed using 40 -60 -diamidino2-phenylindole (DAPI) and propidium iodide (PI) (Sigma-Aldrich, St Louis, MO, USA) following the method of SCHUMANN et al. (26). Exponentially growing cultures of the isolates were treated with 300 lg ml1 of ZnO-NPs for 4 h and untreated cells were used as the control. After treatment, the cells were harvested by centrifugation at 915 g and suspended in autoclaved PBS (pH 7.5). Then, DAPI and PI were added to the cell suspensions at final concentrations of 5 and 10 lg ml1, respectively, and cells were incubated in the dark for at least 20 min at room temperature. Stained cells from the untreated control culture and from culture treated with ZnO-NPs were observed under a fluorescence microscope (Nikon Eclipse 80i; Nikon Co., Tokyo, Tokyo, Japan). Twelve fields for each treatment were counted and the percentage of dead cells was calculated by comparing the numbers of cells stained with PI with those stained with DAPI. Assessment of biofilm formation Quantitative assessment of biofilm inhibition in the presence of ZnO-NPs was performed on 48-well polystyrene plates (Nunc, Roskilde, Denmark), as described by BURTON et al. (27). Aliquots of 500 ll from overnight cultures of Rothia isolates were seeded onto the surface of the wells of the polystyrene plates containing increasing amounts (50, 100, 200, and 300 lg ml1) of ZnO-NPs in sterile BHI broth. Cultures not exposed to ZnO-NPs were used as a control. Plates were incubated at 37°C for 16 h, and then the growth medium was gently removed and the wells were washed three times with 500 ll of PBS to remove unattached cells. The plates were air dried for 15 min and stained with 500 ll of 0.4% Crystal Violet for 15 min, followed by gentle washing three times with 500 ll of PBS

ZnO-NPs for control of oral Rothia sp.

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plasmon resonance in the wavelength range of 200 and 800 nm using a UV-Vis spectrophotometer (Cintra 10e; GBC, Port Melbourne, Vic., Australia) (data not shown).

buffer to remove any unbound dye. Finally, the Crystal Violet retained by the biofilm was dissolved by adding 500 ll of 33% acetic acid. Absorption was read at 620 nm in a microtitre plate reader (Multiskan Ascent; Labsystems, Vantaa, Finland). A similar procedure was used for qualitative assessment of biofilm formation on the surface of artificial acrylic teeth and acrylic denture, except that, after staining and washing, images were recorded using a digital camera (Canon D550; Canon, Tokyo, Japan). In a separate, but similar, set of experiments, bacterial growth and biofilm inhibition were also compared with 100% pure commercially available clove oil with a density of 1.06 g cm3 (Eurostar, LLC, Deira, Dubai, United Arab Emirates). Increasing volumes of 10, 20, 40, 60, and 100 ll of clove oil were added directly to bacterial suspensions, achieving a concentration range of ~10 to 100 mg ml1.

Isolation and characterization of Rothia isolates

Based on the biochemical characteristics, 16S rRNA gene sequence similarities and phylogenic analysis, the two isolates Ora-7 and Ora-16 were identified as R. dentocariosa and R. mucilaginosa, respectively (Table 1). The isolate Ora-7 was found to be Grampositive, catalase positive, and with the capability of gelatin hydrolysis, whereas the isolate Ora-16 was catalase negative and unable to hydrolyze gelatin. Both isolates exhibited glucose and sucrose fermentation and no lactose utilization. The inability of the isolates to grow in the presence of 6% NaCl distinguished them from members of the genera Micrococcus and Staphylococcus (1). Furthermore, the BLASTN and pairwise sequence alignment analyses revealed  99% sequence homology of the 16S rRNA gene of Ora-7 with R. dentocariosa ATCC17931 (accession no. 074568), whereas isolate Ora-16 was most closely related to R. mucilaginosa ATCC25296 (accession no. X87758) with a maximum sequence similarity score of 98%. PCR amplification and sequencing yielded partial nucleotide sequences of 877 and 825 bp for the 16S rRNA genes of the Ora-7 and Ora-16 isolates, respectively, which have been deposited in NCBI GenBank with accession nos. KJ136113 and KJ136114, respectively. The phylogenetic analysis revealed a close relationship between the Ora-7 and Ora-16 isolates and strains of R. dentocariosa and R. mucilaginosa, respectively (Fig. 1). Ora-7 clustered with the strains of R. dentocariosa with a sufficiently high bootstrap value of 972. Ora-16 clustered with the strains of R. mucilaginosa and a branch

Statistical analysis The results presented are the mean  SD of two independent experiments carried out in triplicate. Statistical analysis was performed by the unpaired t-test using GRAPHPAD PRISM version 5.0 (GraphPad Software, La Jolla, CA, USA). The level of statistical significance chosen was P < 0.05.

Results Characteristics of ZnO-NPs

The TEM analysis revealed polygonal and smooth-surface ZnO-NPs with an average size of 35 nm. The XRD pattern of ZnO-NPs reaffirmed that the polycrystalline wurtzite structures had an average size of 35.23 nm, as calculated using Scherrer’s equation. The XRD-based particle size was in agreement with the TEM observations (14). Stability of the ZnO-NPs in aqueous suspension was checked by periodic measurement of surface

Table 1 Biochemical properties of the Rothia isolates (Ora-7 and Ora-16), isolated in this study from the oral cavity, and their comparison with ATCC strains Tests

R. dentocariosa* ATCC 17931T

R. dentocariosa Ora-7

Catalase + Gelatin hydrolysis + TSI slant acid + Growth at 25°C  35°C + 42°C  Growth in nutrient broth containing 0% NaCl + 6% NaCl  Acid from Lactose  Glucose + Sucrose + +, positive; , negative; W, weak. *Data for ATCC strains are taken from DANESHVAR et al. (19).

R. mucilaginosa* ATCC 25296T

R. mucilaginosa Ora-16

+ W/ +

  +

  +

W + +

 + W

+ + +

+ 

+ 

+, W 

 + +

 + +

 + +

4

Khan et al. A

Fig. 1. Phylogenetic tree constructed using the Neighbor joining method showing the positions of the isolates Ora-7 and Ora-16 with standard strains of Rothia dentocariosa and Rothia mucilaginosa. Bootstrap values are shown at the nodes when >700 and are calculated from 1,000 resamplings. Closed circles at the nodes show the branches also found in a maximum-likelihood tree.

support of 1,000 resamplings (Fig. 1), confirming the phylogenetic affiliation of Ora-16 to R. mucilaginosa.

A1

A2

A3

A4

B

B1

B2

B3

B4

Effect of ZnO-NPs on growth of Rothia isolates

The growth-inhibitory effect of ZnO-NPs on Rothia isolates was determined as a reduction in the numbers of viable CFUs. Compared with the control, the reduction of R. mucilaginosa Ora-16 CFUs in BHI broth was 20%, 70%, and 94% with 50, 100 and 200 lg ml1 of ZnO-NPs, respectively (Fig. 2). The R. dentocariosa isolate was found to be invariably more sensitive at all concentrations of ZnO-NPs than was R. mucilaginosa. A 48%, 79%, and 98% reduction in the number of R. dentocariosa Ora-7 CFUs ml1 was observed with 50, 100 and 200 lg ml1 of ZnO-NPs, respectively. The IC50 values of ZnO-NPs were determined to be 53 and 76 lg ml1 for R. dentocariosa and R. mucilaginosa, respectively. The results from the growth-inhibition assay were validated by a viability assay observing live/dead cells using the fluorescent dyes DAPI (Fig. 3A1,A3,B1,B3)

Fig. 2. Biocidal effect of zinc oxide nanoparticles (ZnO-NPs) on the number of colony-forming units of Rothia dentocariosa and Rothia mucilaginosa. Colony-forming unit (CFU) counts are presented as log10 CFUs ml1. Data are presented as mean  SD of two independent experiments performed in triplicate. *P < 0.05, with respect to the untreated control.

C

Fig. 3. Population of dead cells of Rothia dentocariosa Ora-7 and Rothia mucilaginosa Ora-16 after treatment with 300 lg ml1 of zinc oxide nanoparticles (ZnO-NPs). (A1, A3, B1, B3) Total number of cells stained by 40 -60 -diamidino-2phenylindole (DAPI). (A2, A4, B2, B4) Population of dead cells, as determined by propidium iodide (PI) staining. (A4, B4) Population of dead cells after treatment with ZnO-NPs compared with the population of total cells (A3 and B3) in the same fields. (C) Bar chart of the percentage of dead cells (mean  SD of 12 fields) after treatment with ZnO-NPs. CRD, Rothia dentocariosa Ora-7 control; CRM, Rothia mucilaginosa Ora-16 control; RD, Rothia dentocariosa Ora-7; RM, Rothia mucilaginosa Ora-16.

and PI (Fig. 3A2,A4,B2,B4), respectively. The images in Fig. 3A,B show the presence of total cells and dead cells in samples of R. dentocariosa Ora-7 and R. mucilaginosa Ora-16 cells. The population of live cells decreased (Fig. 3A1,A3,B1,B3), whereas that of dead cells increased (Fig. 3A2,A4,B2,B4) substantially upon treatment with 300 lg ml1 of ZnO-NPs (Fig. 3A3, A4,B3,B4). The effects were almost similar for R. dentocariosa Ora-7 and R. mucilaginosa Ora-16 at an identical concentration of ZnO-NPs. The proportion of dead R. dentocariosa Ora-7 cells, as determined by PI staining, was 61  5% of the total treated (with ZnONPs 300 lg ml1) population. The corresponding value for R. mucilaginosa Ora-16 was 42  7% dead cells, and for the controls was 3% (Fig. 3C). For comparison, no significant growth inhibition was

ZnO-NPs for control of oral Rothia sp.

observed with 10 mg ml1 of clove oil, whereas a 34– 53% decrease in the number of CFUs ml1 was observed with 20 mg ml1 of clove oil (data not shown). ZnO-NPs induced inhibition of biofilm formation by Rothia isolates

Treatment of the Rothia isolates with ZnO-NPs inhibited Rothia biofilm formation, in a concentrationdependent manner in the Crystal Violet plate assay, following 16 h of incubation at 37°C (Fig. 4). The inhibition was more pronounced for R. dentocariosa Ora-7 biofilm than for R. mucilaginosa Ora-16 at all test concentrations. At a concentration of 100 lg ml1 of ZnO-NPs, R. dentocariosa Ora-7 biofilm formation on polystyrene was inhibited by 24%, compared with 25% for R. mucilaginosa Ora-16. The corresponding values for R. dentocariosa Ora-7 were 59% and 69% at 200 and 300 lg ml1 of ZnO-NP, respectively. Biofilm inhibition of 47% and 60% was observed at 200 and 300 lg ml1 of ZnO-NP, respectively, for R. mucilaginosa Ora-16. Clove oil showed significant inhibition of the biofilm of the Rothia isolates only at a concentration of 10 mg ml1 or higher (data not shown). Furthermore, the inhibition of biofilm formation by ZnO-NPs was studied on the surface of artificial teeth and dental prostheses, which explicitly showed the biofilm-inhibitory effects of ZnO-NPs on teeth and prostheses coated with R. mucilaginosa Ora-16 and R. dentocariosa Ora-7 (Fig. 5A1,A2,B1,B2). This effect was found to be concentration dependent and was more pronounced for R. dentocariosa Ora-7 (Fig. 5A2, B2) than for R. mucilaginosa Ora-16 (Fig. 5A1,B1).

Discussion Several Rothia spp. are emerging as crucial opportunistic pathogens of the oral cavity. Therefore, minimizing

Fig. 4. Effect of zinc oxide nanoparticles (ZnO-NPs) on biofilm formation by Rothia dentocariosa and Rothia mucilaginosa on the surface of polystyrene plates, measured as Crystal Violet retained by the biofilm. Values presented in the bar graph are mean  SD of two independent experiments performed in triplicate. *P 100 lg ml1 of ZnO-NP for 2–72 h became abnormal in size and displayed cellular shrinkage and detachment from the surface of flasks with a significant decline in the mitochondrial function and lactate dehydrogenase activity. MUSARRAT et al. (37) have also reported weaker toxicity of ZnO-NPs (19.82 nm) at concentrations up to 100 lg ml1 in cultured human lymphocytes. Indeed, the toxicity of NPs varies with the size and shape of the NPs (38). Also, the size and shape of NPs have been shown to affect their antimicrobial activity. For instance, flower-shaped ZnO-NPs, with more sharp edges, have been reported to exhibit higher antimicrobial activity against Escherichia coli and Staphylococcus aureus compared with relatively smoother, rod- and sphere-shaped NPs (39). In general, the important factor that contributes in bacterial growth inhibition could be the release of zinc ions from ZnO-NPs and the generation of reactive oxygen species (31, 40). Leakage of the intracellular contents of bacteria and the disruption of cell walls and cell membranes as a result of

exposure to ZnO-NPs has also been demonstrated (41). Nevertheless, it has been suggested that ZnO is relatively more biocompatible than other NPs with known antimicrobial activity, such as silver NPs, as zinc (but not silver) is a necessary trace element in humans (42). Furthermore, ZnO-NPs, being non-toxic and biocompatible, have been utilized as drug carriers, cosmetics ingredients, and medical filling materials (43, 44). Thus, it is concluded that the engineered ZnO-NPs exhibit substantial antibacterial activity and anti-biofilm inhibition against Rothia isolates. Further studies are warranted to optimize the doses of ZnO-NPs, which are non-toxic to host cells but are effective enough to control bacterial growth and prevent dental problems caused by oral infections. Acknowledgements – Financial support for this study through the National Plan for Sciences and Technology (NPST) Project No. 12-NAN-2490-2, King Saud University, Riyadh is greatly acknowledged. J.M. is grateful to the Visiting Professorship Program (VPP), King Saud University, for overall support to carry out this collaborative research. Conflicts of interest – Authors have no conflict of interests whatsoever.

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Anti-biofilm and antibacterial activities of zinc oxide nanoparticles against the oral opportunistic pathogens Rothia dentocariosa and Rothia mucilaginosa.

Species of the genus Rothia that inhabit the oral cavity have recently been implicated in a number of diseases. To minimize their role in oral infecti...
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