Antibiofilm activity of zinc oxide nanosheets (ZnO NSs) using Nocardiopsis sp. GRG1 (KT235640) against MDR strains of gram negative Proteus mirabilis and Escherichia coli

Accepted Manuscript Title: Antibiofilm activity of zinc oxide nanosheets (ZnO NSs) from Nocardiopsis sp. GRG1 (KT23540) against MDR strains of gram negative Proteus mirabilis and Escherichia coli Authors: Govindan Rajivgandhi, Muthuchamy Maruthupandy, Thillaichidambaram Muneeswaran, Muthusamy Anand, Natesan Manoharan PII: DOI: Reference:

S1359-5113(17)31219-9 https://doi.org/10.1016/j.procbio.2018.01.015 PRBI 11245

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

31-7-2017 21-12-2017 23-1-2018

Please cite this article as: Rajivgandhi Govindan, Maruthupandy Muthuchamy, Muneeswaran Thillaichidambaram, Anand Muthusamy, Manoharan Natesan.Antibiofilm activity of zinc oxide nanosheets (ZnO NSs) from Nocardiopsis sp.GRG1 (KT23540) against MDR strains of gram negative Proteus mirabilis and Escherichia coli.Process Biochemistry https://doi.org/10.1016/j.procbio.2018.01.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antibiofilm activity of zinc oxide nanosheets (ZnO NSs) from Nocardiopsis sp. GRG1 (KT23540) against MDR strains of gram negative Proteus mirabilis and Escherichia coli

Govindan Rajivgandhi1, Muthuchamy Maruthupandy2,3*, Thillaichidambaram Muneeswaran2,

1Department 2

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Muthusamy Anand2 and Natesan Manoharan1

of Marine Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.

Department of Marine & Coastal Studies, School of Energy, Environment and Natural

Resources, Madurai Kamraj University, Madurai, Tamil Nadu, India. 3

School of Chemistry & Chemical Engineering, Anhui Province Key Laboratory of Chemistry

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for Inorganic/Organic Hybrid Functionalized Materials, Anhui University, Hefei, Anhui 230601,

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PR China

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Tel: +86 15855181341 (M), E-mail: [email protected]

Graphical abstract Graphical abstract

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Biomass of Nocardiopsis sp.

ZnO NSs

35 ˚C

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ZnO NSs

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Synthesized ZnO NSs

E. coli Antibiofilm activity

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Zinc nitrate

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Highlights

ZnO NSs were synthesized using biological route of Nocardiopsis sp.

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Screening for biofilm production activity of uropathogens using TCP and CRA assays.

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Investigation for antibiofilm efficiency of ZnO NSs against various MDR strains.

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Morphological damage and cell viability of MDRs by CLSM and SEM analysis.

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Abstract In present investigation, the anti-biofilm activity of zinc oxide nanosheets (ZnO NS) synthesized from actinomycetes of Nocardiopsis sp. GRG1 (KT23540) were analyzed against of P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3

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multidrug resistant (MDR) strains

(KY617770) uropathogens. The synthesized ZnO NSs were characterized functionally using UV–vis absorption spectra, fourier transform infrared (FTIR), X-ray diffraction (XRD) pattern, raman spectra, energy-dispersive X-ray spectroscopy (EDS) and morphologically using scanning electron microscopy (SEM). Detection of biofilm formation was done in biofilm producers and

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non producers were carried out by tissue culture plate method (TCP) and congo red agar assay

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(CRA). The synthesized ZnO NSs showed significant antibacterial and anti-biofilm activity of

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ZnO NSs against MDR strains of P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3

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(KY617770) were determined by discs diffusion and spectroscopy methods using different concentration between 5 and 25 µg/mL and 2 to 25 µg/mL, respectively. The inhibition of

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biofilm formation of MDR stains were morphologically imaged by CLSM and SEM. Our results

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clearly indicate that ZnO NSs could provide a safer alternative to conventional antibiofilm agents

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against MDR strains of P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770). In this study, purpose to produce a novel, cost-effective, eco-friendly actinomycetes mediated

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ZnO NSs synthesis and its antibiofilm activity.

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Keywords: Entophytic actinomycetes; Nocardiopsis sp; ZnO nanosheets; MDR strains; Proteus mirabilis and Escherichia coli; Antibiofilm activity

1.

Introduction The antibiotic resistance among the pathogens is emerging and spread rapidly over the past

decade and it is alarmed that the incidence of MDR development and infection are increasing.

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Treatment of such MDR infected patients are still another problem since the pathogens are almost resistant to all the available drugs and they easily develop resistance to the new drugs also. In addition to MDR, several pathogens form community known as biofilms; around 80 % of the bacterial biomass found as biofilms and said to be the major form of microbial biomass on earth [1]. In addition, Biofilm forming microbes are responsible for more than 80 % of infections [2].

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Biofilms are highly complex assembled in self produced polymer made of extracellular polymeric

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substances (EPS) [3]. This complexity of biofilms makes the bacteria to adhere on live or

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non-living surfaces. Both gram positive and negative microbes form biofilms. Further, EPS

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reduces the diffusion and penetration of antibiotics and the acidic pH, lower oxygen level in core bifilm affects the action of antibiotics. Thus, biofilms assures better survival of the microbial

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community by providing the ability to escape from the action of antibiotics and immune systems

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[4]. Hence, it is necessary to find an alternative way to treat the pathogens without said problems.

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Nanoparticles (NPs) are promising compounds to treat such dreadful MDR pathogens and there is a less possibility of resistance development during treatment with NPs since they directly

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make contact with the bacterial cell wall [5]. Further, traditional antibiotics can be delivered to the site by carrier NPs. Nanomaterials display entirely new or developed properties supported on

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circumstantial characteristics such as size, dispersion, structure and perfect chemical composition. NPs possess unique property compared to their micro sized particles since their high surface area to volume ratio [6]. However, the NPs synthesis may relay on toxic materials, high cost and energy and are highly vulnerable to the environment and human health. Nowadays scientists are very

conscious about the environment and looking for an alternative ways to synthesize materials via green synthesis. One of such eco-friendly method is based on biological organisms including bacteria, fungi and plants that enable the production of chemicals less or without damaging the

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environment [7]. Among the metallic nanoparticles, Zinc Oxide (ZnO) NPS are believed to be less toxic, cheaper and bio compatible and can be synthesized eco-friendly with different morphologies and sizes [8]. Recent studies have shown that ZnO NPs are selectively toxic to bacterial cells and less toxic human cell lines and it disturbs the cell membrane via reactive oxygen species generations [9]. Further, ZnO NPs have been used as surface coating materials and inhibit the

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growth of fungal and bacterial pathogens [10].

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Though, most organisms can be used for the synthesis of NPs by biological route marine

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actinomycetes might display appreciable interest owing to their ability to produce new chemical

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entities with various medical activities. In this study, actinomycetes Nocardiopsis sp. was used for the synthesis of ZnO NPs, as it consists more possibly progressive elements which have been

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reported to possess many biological and chemical properties [11,12]. From the literature, many

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researchers have recently reported the biological synthesis of metal (Ag and Au) nanoparticles

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using actinomycetes [13,14], there are very few reports on actinomycetes mediated synthesis of ZnO NPs [15-17] and this is a first report on ZnO nanosheets (NSs) using actinomycetes.

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Urinary tract infections (UTIs) are one of the most common infectious diseases in humans. It is well known that the occurrence of these infections is much more frequent in

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females than in males [18]. Several pathogens including Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and Serratia marcescens are often implicated in UTIs [19]. Since, the successful establishment of UTIs by several pathogens is mediated through the biofilm mode of growth and targeting biofilm formation may provide better treatment against UTI pathogens. The

present study is focused mainly with the rapid and facile synthesis of ZnO NSs by actinomycetes using zinc nitrate hexahydrate as a substrate. The synthesized ZnO NSs were characterized using UV-DRS, FTIR, XRD, raman spectra, SEM and EDS analysis. Moreover, the antibacterial

BDUMS3 (KY617770) was subsequently examined in details. 2. Materials and methods 2.1. Preparation of actinomycete biomass

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antibiofilm activity against MDR uropathogens of P. mirabilis BDUMS1 (KY617768), E. coli

In the present study marine endophytic actinomycetes of Nocardiopsis sp. GRG 1

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(KT235640) was used for the synthesis of ZnO NSs. The isolation and identification of the

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Nocardiopsis sp. GRG 1 was reported elsewhere [20]. The actinomycete was inoculated into

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starch casein broth medium prepared with 50 % of seawater and incubated at 28 °C for 6 days.

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Culture was centrifuged at 5000 rpm at 4 °C to remove supernatants and the collected mycelium was stored at 4 °C after successive washing with distilled water.

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2.2. Biological synthesis of ZnO NSs

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ZnO NSs were synthesized biologically using the supernatants of actinomycetes of

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Nocardiopsis sp. GRG 1. In brief, about 20 g of the biomass was transferred to 100 mL of 1 mM Zinc nitrate (Zn (NO3)2) solution and kept in shaker incubator at 35 ºC at 120 rpm for 120 h. The

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flask was observed for the visible colour change from yellow to whitish yellow at regular interval. After 24h of incubation, the solution was centrifuged at 5000 rpm for 30 min and the

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pellets were collected. Then, pellets were transferred to a ceramic crucible and heated in furnace at 400 ºC for 3 h. Finally, slightly yellowish white colored product (ZnO NSs) was obtained and used for further studies. 2.2.1. Characterization of ZnO NSs

The absorption characteristic of the synthesized ZnO NSs was recorded in the UV–visible spectrum wavelength range between 200 and 800 nm by using diffuse reflectance spectral (DRS) accessory equipped in a UV–vis spectrophotometer (UV 2500 Shimadzu). Fourier transform

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infrared (FTIR) spectrum was recorded between 4000 and 400 cm−1 using Shimadzu 8201 PC. X-ray diffraction (XRD) pattern of the ZnO NSs was recorded between the 2θ range of 10 and 80° in a X-ray diffractometer (XPERT-PRO), operated at a voltage of 40 kV and a current of 30 mA with Cu okα radiation (λ= 1.54060 Å). Raman spectra were registered from 100 to 1000 cm−1 on a Horiba Jobin–Yvon LabRAM HR 800 using a632.8 nm He-Ne laser at a power of 0.5

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mW. The powder samples were dispersed onto a microscope slide and spectra were collected.

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For surface morphology SEM images have been recorded in SU-70 (ModelL-hi-0028-0001)

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operated at an applied voltage of 10 KV and energy-dispersive X-ray spectroscopy (EDS) was

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also studied for identification of ZnO NSs atomic percentage. 2.3. Bacterial pathogens maintenance

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The multi drug resistant uropathogens of P. mirabilis BDUMS 1 (KY617768) and E.coli

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BDUMS 3 (KY617770) were procured from the unit of Microbiology & Pharmacology,

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Department of Marine Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. All strains have the ability to develop resistance against almost all clinical drugs was reported by

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previously Rajivgandhi et al. [21] and these strains were stored at -20 ºC and used for this study.

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2.3.1. Identification of resistance by HEXA UTI 5 disc method The drug resistance among the pathogens were also confirmed by Kirby-Bauer disc

diffusion method as per CLSI guidelines using the Hexa UTI 5 disc method containing antibiotics ciprofloxacin (Cip-5 mcg), ampicillin (AMP-10 mcg), amoxyclav (AMC-30 mcg), norfloxacin (NX-10 mcg), nitrofurantoin (NIT-300 mcg), co-Trimoxazole (Cot-25 mcg) [22].

2.4. Antibacterial activity by ZnO NSs The antibacterial activity of the synthesized ZnO NSs against P. mirabilis BDUMS1 (KY617768), E. coli BDUMS3 (KY617770) was done by Kirby buyer disc diffusion assay. The

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overnight grown bacterial cells (~10-4) were spread over the muller hinton agar (MHA) plates to produce confluent area of the bacterial growth. The 6 mm of sterile discs (Himedia, India) were impregnated with various concentrations of ZnO NSs (5, 10, 15, 20, and 25 µg/mL) and dried. The sterile discs were placed on the swapped MHA plates and incubated at 37 ºC for 24 hrs. After incubation the zone of inhibition around the discs in diameter (mm) was measured. The values are

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the averages of the triplicates of the test results.

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2.5. Detection of biofilm formation

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Strains of P. mirabilis BDUMS1 (KY617768), E. coli BDUMS3 (KY617770) and two

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reference strains of non biofilm producers P. mirabilis MTCC 425 and E. coli MTCC 443 were tested for their biofilm formation using tissue culture plate method (TCP) and congo red agar assay

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(CRA) assays

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2.5.1. Tissue culture plate method (TCP)

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The biofilm formation TCP assay was carried out using 96-well polystyrene plate method (Christensen, et al., 1985). Briefly, 150 µL of 10-5 cfu/mL of all strains were inoculated into the test

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tube containing fresh tryptic soya broth and incubated at 37 ºC for 24 h. After incubation, 50 µL of the cultures were seeded into the wells of microtitre plate (Himedia Laboratory, India) containing

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100 µL of tryptic soya broth with 1 % glucose and the sterile broth served as a control. After 24 h of incubation at 37 ºC, the plates were washed with 0.2 mL of phosphate buffered saline (PBS) to detach the free planktonic cells. Adherent bacteria on the plate were stained with 0.4 % crystal violet solution (w/v) for 10 min. Subsequently the excess dye was rinsed off by washing twice with

deionized water and then allowed to dry. Finally, 1 mL of absolute ethanol was added to each well. The optical density was determined at 590 nm (Mathur et al., 2006) in microtiter reader. The presence and absence of biofilm formation were considered based on the OD value (Table 1). The

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experiment was performed in triplicates. Percentage of inhibition= (Control OD570nm-Test OD570nm) / Control OD570nm X 100 Adherence

Mean value of OD

High

Strong

>0.240

Moderate

Moderate

0.120-0.240

Non/Weak

Non/Weak

<0.120

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Biofilm formation

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2.5.2. Congo red agar assay (CRA)

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The biofilm production of the strains was further confirmed by CRA method [23]. The

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overnight cultures were inoculated with CRA medium and incubated for 24 h at 37 °C. Black coloration around the colonies was considered as positive biofilm producer, whilst pink coloured

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colonies were considered as non biofilm producers.

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2.6. Detection of antibiofilm activity

The biofilm inhibition effect of the ZnO NSs was tested against strong biofilm forming

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strains such as P. mirabilis BDUMS 1 (KY617768) and E. coli BDUMS 3 (KY617770) in 24 well

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polyvinyl chloride microtiter plates with some modifications of Stepanovic et al. [24]. Briefly, overnight grown cultures (100 µL) were seeded in wells containing 1 mL of fresh TSB medium

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and various concentrations (2 - 25 µg/mL) of ZnO NSs were added to the wells and incubated at 37 °C for 24 h. The wells without ZnO NSs addition served as control and medium alone served as a blank. After incubation, the cells were removed and rinsed thoroughly with distilled water and air dried. The plates were stained with 600 µL of 0.1% crystal violet for 30 min and the excess dye was removed by washed twice with deionized water and allowed to dry. Finally, 1 mL of dimethyl

sulphoxide was added in each well and the OD value was determined at 570 nm. The percentage of inhibition was calculated using the following formula: Percentage of Inhibition = [Control OD 570 nm - Test OD 570 nm / Control OD 570nm] X 100

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2.7. ZnO NSs treated MDR strains morphological analysis 2.7.1. Light microscope

For the visualization under light microscope, the MDR strains were allowed to grow on glass pieces (1x1 cm) placed in test tube supplemented with and without ZnO NSs at a concentration of 20 µg/mL and incubated for 24 h at 37 °C. The glass pieces were then washed

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with PBS and stained with 0.4 % crystal violet. The stained glass pieces were placed on the

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slides and directly visualized under light microscope at 40 X magnification [25].

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2.7.2. Confocal laser scanning microscopy (CLSM)

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The inhibition of biofilm formations were analyzed by CLSM. The 24 h lag phase MDR bacterial cultures were inoculated with fresh sterilized tryptic soy broth and the cells were treated

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with ZnO NSs at the biofilm inhibition concentration (BIC) of 20 µg/mL. The plates were

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incubated at 37 °C for 24 hrs. After incubation, the cells were collected by centrifugation at 5000

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rpm for 10 min at 4 °C. After centrifugation, the supernatant were discarded and the pellet were collected and washed three times with PBS. The cells were stained with 100 µL of florescent dye

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acridine 10 mg in 10 mL of PBS, pH=7.4) for 15 min under dark condition. The cells were rinsed with PBS and stained cells were analyzed under CLSM (The 488 nm argon laser and a 500-640 nm

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band pass emission filter). The untreated cells (control) were also stained and observed under CLSM [26]. 2.7.3. Scanning electron microscopy (SEM)

The membrane integrity of the treated and untreated P. mirabilis BDUMS 1 (KY617768) and E. coli BDUMS 3 (KY617770) were visualized under SEM. The treated and untreated cells after biofilm inhibition assay were washed and resuspended in10 mM sodium phosphate buffer

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(pH 7.4) and fixed with an equal volume of 4 % glutaraldehyde. Fixed cells were vacuum filtered onto a 0.1 mm polycarbonate membrane filters and dehydrated through a graded series of ethanol (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 %). The filters were then dried and mounted on aluminum specimen support and coated with 15 nm thickness of gold-palladium metal (60:40 alloys). Samples were examined on a Cambridge Stereo scan 200 SEM using an accelerating

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voltage of 20 kV was performed [27].

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3. Results and Discussions

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3.1. ZnO NSs characterization

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UV–vis diffused reflectance spectrum (DRS) of ZnO NSs was shown in Fig. 1a. The DRS spectrum suggested an individual broad band at 385 nm that is blue shifted with respect to the ZnO

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absorption edge. No other peaks were observed in the DRS spectrum, indicated that the ZnO NSs

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synthesized in the present study was highly high pure. This blue shift in the absorption maximum

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clearly displays the quantum confinement property of NSs [28]. Within the quantum confinement range, the band gap of the particles raises ensuing in the shift of absorption edge to lessen

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wavelength. The observed absorption band is due to the transition from the valence band to the

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conduction band and concurs with the reported literature for ZnO NSs [29-30]. FTIR spectrum of the synthesized ZnO NSs was shown in Fig. 1b. The peak at 532 cm−1 is

the typical absorption peak of Zn-O and the extensive absorption peak at 3480 cm−1 is due to −OH stretching vibration of hydrogen bond indicated the continuation of hydroxyl group [30]. The occurrence of peak at 1516 and1328 cm−1 is attributed to N−O stretching vibration mode of nitro

aromatic compounds. The band at 875 cm−1 is due to C−H stretching of aromatic meta-disub of benzene. The secondary metabolites of actinomycetes biomass acted as reducing, capping agent and space the ZnO NSs formed from aggregation. The presence of macrolides, polyene antibiotics,

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aminoglycosides and anthracyclines are considered to play a main role in response to Zn ions and stabilize the ZnO NSs [31]. Merely, the possible mechanism is still uncertain and needs extra exploration.

1519 1328

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b

3480

875

300

400

500

700

532 3000

2000

1000 -1

Wavenumber (cm )

UV-Vis (DRS) spectra (a) and FTIR spectra (b) of ZnO NSs.

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Fig. 1.

600

4000

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Wavelength (nm)

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Absorbance (a.u.)

Transmittance % (a.u.)

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The crystal phases and crystallinity of the synthesized ZnO NSs were analyzed by XRD

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pattern, and shown in Fig. 2a. As seen in Fig. 2a, the peaks at 2θ values of 31.73º, 34.40º, 36.21º, 47.49º, 56.52º, 62.80º, 67.86º and 68.99º which were corresponded to the plans of (100), (200),

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(101), (102), (110), (103),(200),(112) and (201) respectively. The observed lattice values are in

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well agreement with the standard spectrum of sheet like structure with (JCPDS card number 036-1451 [32]. As, there was no other distinctive peaks detected, indicated the high-purity of formed ZnO NSs. A broad spectrum exhibited by the sample before calcination indicates that the dried samples demonstrated an amorphous behaviour although physically appeared to behave like crystalline materials. For the calcined samples at 500 ºC and above, the spectrum shows

sharper and narrower diffraction peaks, implying that the crystalline ZnO NSs were formed [33]. Raman spectrum of ZnO NSs was given in Fig. 2b. The ZnO NSs demonstrated the regular Raman manner and they were ascertained at 330 (second-order vibration related to the E2(high)-E2(low)),

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433 (E2(high)), and 582 cm－1 (E1(LO)) (Chen et al., 2008, Gayathri et al., 2014). From the Raman scattering, in the backscattering geometry, ZnO NSs with the sheet like structure should display phonons of the symmetry E2(high) at 433 cm － 1 [34]. Consequently, the E2 high) condition determined in the Raman spectrum of the synthesized ZnO NSs was confirmed. Hence, the ZnO nano structure formation of sheet like morphology was confirmed through Raman spectral

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2 (degree)

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E2(high)

E2(low)

70

200

E2(LO)

E2(high)-E2(low)

(112) (201)

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(200)

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(103)

(110)

(102) 40

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30

b

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JCPDS No: 036-1451

Raman intensity (a.u.)

(101)

(100) (002)

Indensity (a.u.)

a

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analysis.

400

600

800

Wavenumber cm-1

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Fig. 2. XRD pattern (a) and raman spectra (b) of ZnO NSs. The surface morphology of ZnO NSs was demonstrated using scanning electron microscopy

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(SEM) and their different magnifications were displayed in Fig. 3a-d. Fig. 3a-d reveal that the product has a sheet like morphology with a thin edges and irregular morphology structure were shown and size range of ZnO NSs between 500 nm to few micron [35,36]. However, ZnO NSs product gradually started to break down and overlapped in a proportional relation with the increment of calcination temperature as shown in Fig. 3a-d. The elemental composition of the

ZnO NSs sample was also determined using energy dispersive X-ray (EDS) spectroscopy and illustrated in Fig. 3e. It was evident that the Zn and O elements were present in the prepared sample as evident by their respective peaks. The recorded atomic percentages of Zn and O were

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51.45 % and 48.55 %, respectively. The recorded elemental weight percentage Zn was 81.24 and O was 18.76. This EDS result indicated that the synthesized final product is pure ZnO NSs without any contamination.

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b

5 µM

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10 µM

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1 µM

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2 µM

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Fig. 3. Different magnification of SEM images (a - d) and EDS spectrum (e) of ZnO NSs. 3.2. Detection of MDR uropathogens Based on the zone of inhibition, the highest degree of resistance was observed in P. mirabilis BDUMS1 (KY617768) plate and it was resistant to all HEXA disc antibiotics. For E. coli

BDUMS3 (KY617770), the observed zone of inhibition with ciprofloxacin was 10 mm and with norfloxacin was 5 mm and no zone of inhibition was observed with antibiotics (Fig. 4a,b). It is clear that the tested uropathogens were developed resistance to almost all the antibiotics used in

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the present study. The Enterobacteriaceae E. coli, P. mirabilis, K. pneumoniae, Acinetobacter sp and Pseudomonas aeruginosa were alarming degrees of antimicrobial resistance and are associated with high mortality and morbidity due to scarcity of effective antibiotics [37]. The prevalence of resistance mechanisms in gram negative bacteria was responsible for the production of biofilm formation that was a reason for MDR. The resistance of both P. mirabilis BDUMS1

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(KY617768) and E. coli BDUMS3 (KY617770) to antibiotics were confirmed by CLSI and Hexa

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Disc method. The results from the both method showed that the pathogens developed resistance

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almost all the pathogens and confirmed that the pathogens were multi drug resistance strains.

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Similar results were reported by Yadhav et al. [38] that the Hexa UTI disc of amikacin (30 μg), ciprofloxacin (5 μg), cotrimoxazole (23.75 μg sulfamethoxazole/1.25 μg trimethoprim), nalidixic

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acid (30 μg), nitrofurantoin (300 μg), norfloxacin (10 μg), and ofloxacin (5 μg) could not inhibited

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the growth of uropathogens tested. Further, the gram negative uropathogens exhibited multi drug

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resistant, and an analysis of the risk factors showed that previous use of fluoroquinolones and various drugs were the strongest determinants of the acquisition of ciprofloxacin resistance by P.

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mirabilis [39].

a

b

Fig. 4. Detection of MDR strains of P. mirabilis BDUMS1 (KY617768) (a) and E.coli BDUMS3 (KY617770) (b) using Hexa disc method 3.3. Antibacterial activity by ZnO NSs The maximum zone of inhibition of 27 ± 0.5 and 24 ± 0.5 mm were observed against P.

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mirabilis BDUMS 1 (KY617768) and E. coli BDUMS 3 (KY617770) at the concentration of 25 µg/mL of ZnO NSs (Fig. 5a,b). Whereas, minimum zone of inhibition 10 ± 0.5 and 14 ± 0.5 mm was observed at the concentration of 5 µg/mL against P. mirabilis BDUMS 1 (KY617768) and E. coli BDUMS 3 (KY617770). However, the Nocardiopsis sp. (KT235640) extract also showed inhibition effect on P. mirabilis BDUMS 1 (KY617768) and E. coli BDUMS 3 (KY617770) with

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inhibition zone of 10 ± 0.5 mm and 5 ± 0.5 mm and no zone of inhibition observed with DMSO

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(Fig. 5 a,b). it can be concluded that the ZnO NSs can inhibit th growth of MDR uropathogens P.

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mirabilis BDUMS1, E. coli BDUMS2 and the excellent antibacterial activity at the concentration

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of 25 µg/mL (Fig. 5c) and it was compare to actinomycete extract (Fig.5d).

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Our result were in contrast with earlier study of Al-Hazmi et al. [40], stated that

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chemically synthesized ZnO NSs could inhibit the growth of food spoiling pathogens such as Bacillus sp. and E. coli at the highest concentration of 600 µg/mL. Whereas, at lower

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concentrations no growth inhibition was observed. The study of Ansari et al. [41] indicated that

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the increased concentration of ZnO NPs did not show significant effect on the zone size. Recently, Balraj et al. [16] reported that the ZnO NSs from marine actinomycetes Streptomyces

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sp. showed antimicrobial activity against various types of bacteria and anti-cancer activity against A549 lung cancer cells at very lowest concentration. Santhoshkumar et al. [30] was also rational with us and reported that the chemically synthesized ZnO NSs against S. aureus, E. coli, P. aeruginosa and B. subtilis were found to be 23, 25, 19 and 24 mm at the low concentration (100 µg/mL). The similar report was also evident by Ansari et al. [42], that the inhibition ability

of biologically synthesized ZnO NSs against MDR gram negative bacteria were showed the maximum inhibition zone of 22 and 20 mm at 100 µg/mL and minimum zone of 8 and 9 mm at 500 µg/mL against E. coli and K. pnumniae respectively. Hence, our result revealed that the

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biologically synthesized ZnO NSs was better than chemically synthesized NSs and the synthesized NSs was very effective against gram negative uropathogens at the minimum concentration of 25 µg/mL. 3.4. Quantification of biofilm formation

The biofilm forming ability of P. mirabilis BDUMS 1 (KY617768) and E. coli BDUMS 3

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(KY617770) were examined by the crystal violet staining assay. After 24 hrs incubation, the OD

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values of 0.465, 0.640 were observed for P. mirabilis BDUMS1 (KY617768) and 0.640 for E.

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coli BDUMS3 (KY617770) respectively. However, the control strains of P. mirabilis MTCC 425

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and E. coli MTCC 232 did not show biofilm formation indicated by the OD values of 0.114, 0.112. The result proved that the two test strains were able to produce strong biofilm (Table 1).

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Table 1. Identification of biofilm formation pathogens.

OD value

Biofilm Production

P. mirabilis BDUMS1 (KY617768)

0.465

Strong

0.640

Strong

0.114

Weak

0.112

Weak

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Name of the Isolates

E. coli BDUMS3 (KY617770)

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P.mirabilis MTCC 425

A

E.coli MTCC 232

a Act. extract

b

Act. extract 25 µg

Contro l

5 µg

control

25 µg

24

8

P. mirabilis BDUMS 1 (KY617768) E. coli BDUMS 3 (KY617770)

6

N

16

10

d

U

c

A

8

0 0

5

10

15

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Zone of inhibition (mm)

P. mirabilis BDUMS 1 (KY617768) E. coli BDUMS 3 (KY617770)

Zone of inhibition (mm)

12

32

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5 µg

20

25

2 0 0

5

10

15

20

25

30

Concentration of Act. extract(g)

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Concentration of ZnO NSs(g)

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Fig. 5. Antibacterial activity of low and high concentration of ZnO NSs against P. mirabilis BDUMS1 (KY617768) (a) E. coli BDUMS3 (KY617770) (b) and graph determination of antibacterial effect in various concentrations of ZnO NSs (c) and actinomycetes extract (d) against P. mirabilis BDUMS1 (KY617768) and E.coli BDUMS3 (KY617770). The biofilm formation was further confirmed congo red agar plate using CRA method.

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After 24 h of incubation the plates were observed for the changes in the colour. Both strains were produce black colour colonies (Fig. 6a,b) on the congo red agar plates indicated the biofilm

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forming ability of the isolates . Whereas, the control strains formed pink color colonies (Fig. 6c,d) indicated inability of biofilm formation. The CRA method was also supportive evidence for production of biofilm formation. The significant result was documented by Rajivgandhi et al. [21] and reported that the strong biofilm forming gram negative bacteria was identified from urinary

tract infection by using TCP and CRA method. Recently, Neupane et al. [23] reported that the antimicrobial resistance of ESBL producing uropathogens P. mirabilis and E. coli could produce biofilm formation in CRA plates and leads to kidney stone. The antimicrobial resistance of gram

showed (80%) by correlation.

a

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d a

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c a

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ab

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negative uropathogens were produced more biofilm formation was also exhibited and result was

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Fig. 6. Conformation of biofilm formation using congo red agar plate method indicated the biofilm positive black color colonies of P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770) (a,b) and biofilm negative pink color colonies of P. mirabilis MTCC 425 and E. coli MTCC 232 (c,d).

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3.5. Biofilm inhibition assay In-vitro inhibition ability of dose dependent ZnO NSs against intense biofilm producers of

two gram negative uropathogens P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770) was determined by crystal violet binding assay. After 24 h treatment with ZnO NSs at the concentration of 20 µg/mL, 92 and 90 % of the growth of P. mirabilis BDUMS 1

(KY617768) and E. coli BDUMS 3 (KY617770) were inhibited respectively compared to the negative control. In the present study, the lowest concentration 20 µg/mL of ZnO NSs was successfully inhibited the biofilm formation in two strains than the highest concentration (Fig. 7).

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Hence, 20 µg/mL of ZnO NSs was selected as a biofilm inhibition concentration (BIC) for both strains. The bacterial adherence plate assay for biofilm mass examined with 20 µg/mL were more effective in collapse of the biofilm colonization and cell bundles of biofilm architecture of P. mirabilis and E. coli were observed. Patil et al. [43] reported that the viability of biofilm forming P. aeruginosa was decreased to 0 % at 125 g/mL of ZnO NSs. Jesline et al. [44] reported that that

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the maximum zone of inhibition against strong biofilm producing MRSA isolate was 16 and 17

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mm at 500 µg/mL and a minimum zone of inhibition of 12 and 14 mm at 100 µg/mL were

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observed. The recent study was demonstrated by Dwived et al. [45] stated that the ZnO NPs

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inhibit S. mutant on oral surface at the concentration of 50 µg/mL. Surprisingly, the biofilm inhibition activity was showed at concentration of ZnO NSs slightly lower than those that affected

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cell viability. However, the increasing concentration of ZnO NSs did not suggest any considerable

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differences between these two uropathogens. In the present study revealed that, the biofilm

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forming uropathogens were more sensitive to ZnO NSs than was cell death. This evident exhibited that various signalling mechanisms could be play a role in cell survive and biofilm

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formation.

80 60 40 20 0 0

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12

16

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Concentration of ZnO NSs(g)

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Bioflim inhibtion assay (%)

P. mirabilis BDUMS 1 (KY617768) E. coli BDUMS 3 (KY617770)

100

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Fig. 7. Biofilm inhibition assay using different concetration of ZnO NSs against P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770).

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3.6. Microscopic observations

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3.6.1. Light microscope

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The ZnO NSs treated and untreated cells were observed under light microscope for the

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detection of biofilm inhibition. The images of the untreated slides showed clear morphology with

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matured biofilm of P. mirabilis BDUMS 1 (KY617768) and E. coli BDUMS 2 (KY617770) Fig. 8a,b. Whereas, the test pathogens showed detached cells of weak biofilm formation at the

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concentration of 20 μg/mL of ZnO NSs (Fig. 8c,d).

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Fig. 8. Light microscopic observation of (a, c) controls (P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770) ), (b) images shows the ZnO NSs treated P. mirabilis BDUMS1 (KY617768) strain, (d) images represents the ZnO NSs treated E. coli BDUMS3 (KY617770).

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3.6.2. Confocal laser scanning microscope (CLSM) The effect of ZnO NSs on the biofilm formation of architecture and surface topography of

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P. mirabilis BDUMS1 (KY617768) and E.coli BDUMS3 (KY617770) was analyzed CLSM and the images were presented in Fig. 9. From the results it was evident that the presences of ZnO NSs

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have significant effect on the biofilm formation of both pathogens. In addition, NSs were entered

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into the cells however, isolate P. mirabilis significantly uptake more ZnO NSs than E. coli. Further, the ZnO NSs treated cells were highly dispersed than the untreated cells at the concentration of 20 µg/mL (Fig. 9b,d). Whereas, the intense adherent ability and depicted well, smooth membrane morphology on the glass pieces of control slides were observed in Fig. 9a,c.

For assessing the ZnO NSs uptake by P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770), the confocal images showed that ZnO NSs treated P. mirabilis BDUMS1 (KY617768) and BDUMS3 (KY617770) cells were successfully uptake the ZnO NSs. The uptake

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was even better in than in E. coli strains. The CLSM exhibited drastically distributed colonies in the biofilm formation were observed at the BIC concentration of the dispersion of colonies observed with ZnO NSs treated cells suggested that the synthesized NSs have significant effect on the biofilm formation of the MDR pathogens. The slides were stained with AO and observed and the results of the CLSM images showed that the bacterial biofilm architecture was frequently lost

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due to the interaction of cells upon treatment of NSs. Hence, the result proved, our synthesized

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ZnO NSs has the potential antibiofilm effect.

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Fig. 9. CLSM observation of (a, c) controls (P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770)), (b) images shows the ZnO NSs treated P. mirabilis BDUMS1 (KY617768), (d) images represents the ZnO NSs treated E. coli BDUMS3 (KY617770).

The fluorescent microscopic analysis of the cells treated with ZnO NSs revealed that the biofilm architecture was disintegrated and the cells were scattered thus very few cell aggregates were formed. Notably, less adherent cells in the aggregates were found after the exposure to ZnO

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NSs. Our results were supported by the significant result documented by Patil et al. [43]. The results revealed that the ZnO NSs were highly active against the biofilm formation of P. aeruginosa. And most of the cells were observed as detached forms that resulted in the drastically damaged the surface morphology of the biofilm were the results of the post exposure to ZnO NSs Recently, Ashajyoth et al. [46] reported that the biologically synthesized ZnO NPs were

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highly active at the concentration of 100 µg/mL against biofilm forming MDR bacteria. Further,

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the ZnO NSs inhibited the biofilm formation at its irreversible adhesion stage (also known as

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initial stage) and the damage of bacterial membrane was clearly observed with detached cells by

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CLSM. Surprisingly, The MIC value was most responsible for inhibition of biofilm formation in primary stage. Our result was accordance with earliest study of Khan et al. [47], the ZnO NPs

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inhibited the biofilm formation in oral opportunistic pathogens R. mucilaginosa Ora-16 and R.

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dentocariosa Ora-7 at the concentration of 200 µg/mL and revealed that the inhibition was dose

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dependent.

3.6.3. Scanning electron microscope (SEM)

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The morphological alterations of the biofilm forming uropathogens were analysed by SEM. After dehydration, the smooth structures of spherical and rod shaped morphology were

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clearly observed in the untreated (control) sample of P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770) (Fig. 10a,c). When compared to control, the attachments of ZnO NSs to the surface of MDR bacterial cell images were exhibited with retaliated effect of morphological damage in the treated strains (Fig. 10b,d). After treatment, cell membrane was agitated in result

with alteration in morphological emphasize in cell wall death/leakage of cellular content. Hence the treated uropathogen morphology was compromised and the inconsistency of cellular content leakage was observed in Fig. 10b,d. Therefore, the ZnO NSs were more evident as a potential cell

a

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2 µM

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2 µM

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wall damaging compound of the MDR bacteria strains.

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2 µM

2 µM

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Fig.10. SEM analysis of (a, c) controls (P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770)), (b) images shows the ZnO NSs treated P. mirabilis BDUMS1 (KY617768) strain, (d) images represents the ZnO treated E. coli BDUMS3 (KY617770) strain.

The formation of wrinkle surface and loss of membrane integrity were observed in the

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surface membrane morphology of ZnO NSs treated P. mirabilis BDUMS1 (KY617768) and E. coli BDUMS3 (KY617770) at 20 µg/mL in SEM analysis. The results revealed that the lowest concentration of ZnO NSs is very effective against MDR pathogens and biofilm inhibition. The rational study done by Patil et al. [43] revealed that the observation of smooth layer of matrix with an under covered uniform cells were the significant effect of the synthesized ZnO NSs against

biofilm forming P. aeruginosa. The impedance of biofilm in P. aeruginosa was inhibited by ZnO NSs was also clearly showed and supported by topographical observation under FESEM. Recently Manzoor et al. [27] reported that the exposure to ZnO NPs for 4 h, resulted in irregular cell

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surface of E. coli and leakage of cellular components of S. aureus. Both of the survival assay and SEM results revealed that the ZnO NPs not only induced alteration in cellular morphology but also cause a lethal effect on the tested bacteria. The supportive results was evident and the loss in viability were correlated to impairment of cell membrane integrity resulted in the damage of the cell walls of the strains Mu et al. [48].

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4. Conclusion

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In the present study, ZnO NSs was successfully synthesized by extracellular biological

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method using marine actinomycetes Nocardiopsis sp.GRG1 (KT235640) at room temperature.

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And the synthesized ZnO NSs was highly stable without addition of reducing and stabilizing

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agents. The present study provided an eco-friendly, cost effective and simple approach for ZnO

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NSs synthesis. The FTIR peak at 3480 cm–1 corresponding to O–H stretching indicated the presence of secondary metabolite that accounted for the reduction and stabilization of ZnO NSs.

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Furthermore, the synthesized ZnO NSs heighten the therapeutic effectiveness and strengthen the

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medicinal values of Nocardiopsis sp. Therefore, our results are promising and confirmed to be an essential measure in this path as it decreases the encumbrance of multidrug resistant

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uropathogens.

Acknowledgement The authors are grateful to the University with Potential for Excellence (UPE) Scheme, Madurai Kamaraj University for generously providing UV, FTIR and XRD instruments facility. The authors express their thanks to Dr. G. Maduraiveeran, Postdoctoral Associate, Lakehead University, Canada for SEM analysis and Central Instrument Facility, Bharathidasan University for CSLM analysis.

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