Biotechnological

Communication

Biosci. Biotech. Res. Comm. 10(3): 380-385 (2017)

Antibacterial activity of ZrO

against metallo

beta-lactamase and bio lm producing

carcinogenic

Pseudomonas aeruginosa

Rajasekar S., Vijayalakshmi S. and A. Mohankumar*

Division of Microbial Technology Post Graduate and Research Department of Zoology Chikkanna Government

Arts College, Tirupur-641602, Tamilnadu, India

ABSTRACT

Pseudomonas aeruginosa is frequent pathogen associated with hospital acquired infections exhibiting intrinsic resist-

ance to numerous antibiotics. A total of 80 isolates of Pseudomonas spp. were isolated from blood cancer patient

from three tertiary hospitals in and around Tirupur and Erode (Dt). Metallo beta-lactamase and bio lm production

is the most worrisome resistant mechanisms observed in P. aeruginosa. Emergence of antimicrobial resistance by

pathogenic bacteria is a major health problem in recent years. The bio lm and Metallo beta-lactamase production

was tested by combined disc test and tissue culture plate method. Electrophoretic analysis of the plasmid DNA pre-

pared was carried out by agarose gel electrophoresis on 0.7%. Nineteen carcinogenic isolates which was showed more

than 50% resistance against tested antibiotics were selected for plasmid isolation. Recurrently, ZrO

nanoparticles

comprises of well-known inhibitory and bactericidal effects. The nanoparticles were obtained and tested against 10

Metallo beta-lactamase and bio lm producing carcinogenic isolates. The nanoparticles showed appreciable activity at

all tested concentrations (0.2, 0.4 and 0.6 mg/ml). Thus, it is concluded that the present study designed to determine

the ef cacy of ZrO

may serve as a promising antibacterial agent against cancer causing Pseudomonas aeruginosa.

380

ARTICLE INFORMATION:

*Corresponding Author: moniver65@gmail.com

Received 27

July, 2017

Accepted after revision 21

Sep, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF: 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/10.3/7

INTRODUCTION

Pseudomonas aeruginosa is one of the most important

opportunistic bacteria, causing a wide variety of infec-

tions especially in immune compromised hosts such

as burn patients, patients suffering from respiratory

diseases like cystic  brosis, and cancer chemotherapy

patients (Govan and Deretic, 1996). It is gram negative

short rod belong to family Pseudomonacaeae. Infections

with P. aeruginosa is occupy very important position as

of greatest concern in critically ill and immune compro-

mised patients who have been hospitalized for extended

Rajasekar S., Vijayalakshmi S. and A. Mohankumar

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ANTIBACTERIAL ACTIVITY OF ZRO

AGAINST METALLO BETA-LACTAMASE 381

periods of time and have received broad-spectrum anti-

microbial therapy or cancer chemotherapy (Pollack,

2000, Ae Mftah et al., 2015).

The pathogenic cancer causing Pseudo bacterial life

includes stages where the cells are associated and form a

bio lm on a surface (e.g. Costerton et al., 1995). The for-

mation of these surface communities and their inherent

resistance to antimicrobial agents are the cause of many

persistent and chronic infections (Costerton et al., 1999).

Nowadays the P. aeruginosa presents a serious ther-

apeutic challenge for treatment of both community-

acquired and nosocomial infections, and selection of the

appropriate antibiotic to initiate therapy is essential to

optimizing the clinical outcome (Mavroidi et al., 2005).

Unfortunately, selection of the most appropriate anti-

biotic is complicated by the ability of P. aeruginosa to

develop resistance to multiple classes of antibacterial

agents, even during the course of treating an infection.

Further acquired resistance is also reported by the pro-

duction of plasmid mediated AmpC beta ()–lactamase,

Extended Spectrum Beta ()–Lactamase (ESBL) and met-

allo beta ()–lactamase (MBL) enzymes.

Currently, the nanoparticles are increasingly recog-

nized for their utility in biological applications includ-

ing nano medicine (mariappan et al., 2011). The ongoing

worldwide nanotechnology revolution is predicted to

impact several areas of biomedical research and other

science and engineering applications. Nanoparticles-

assisted drug delivery, cell imaging, and cancer therapy

are important biomedical applications of nanotechnol-

ogy (Hua Wang et al., 2011).

Despite their potential biomedical applications, very

few studies have reported on the role of zirconia nano-

particles as anticancer materials. As far as the authors

know, this is the  rst report of the in vitro anticancer

effect of sulphated zirconia nanoparticles against three

cancer cell lines. Speci cally, the toxicity of sulphated

zirconia nanopar ticles against human colon cancer

HT29, human breast cancer MCF-7 and human liver

cancer HepG2 cell lines was assessed, showing promis-

ing results (Ae Mftah et al., 2015).

Moreover, it is reported here that these novel

nanopar ticles hold promise not just for anticancer appli-

cations but also for anti-infection applications. The

steady increase in the antimicrobial resistance of micro-

organisms represents a great public health concern. This

requires the search for new unconventional antimicro-

bial agents. Nanotechnology provides promising mate-

rials to  ght infectious diseases such as nanoparticles

with antimicrobial activities. So hence the present study

has made an attempt to point out the antimicrobial

properties of the ZrO

nanoparticles were determined

using the agar diffusion method against cancer causing

Pseudomonas aeruginosa.

MATERIALS AND METHODS

A total of 100 clinical cancer blood samples were col-

lected from the jugular vein with a sterile syringe of

different age group of blood cancer patients in different

clinics and PHC (Primary Health Centre) from in and

around Tirupur and Erode District. 80 isolates of Pseu-

domonas aeruginosa were isolated from the samples. Col-

ony morphology was reported as non-mucoid or mucoid

and the isolated colonies were subjected to standard

biochemical tests and 16S r-DNA gene sequencing for

identi cation of Pseudomonas aeruginosa. Finally all

isolates were kept at – 20

C in media containing 8%

dimethyl sulfoxide (DMSO) until use.

The qualitative and quantitative analyzes of the bio-

 lm produced were performed of clinical isolates of

P. aeruginosa according to the protocol described by

(Xicohtencatl - Cortes et al. 2015). Clinical strains of

P. aeruginosa were incubated for growth in trypticase-

soy broth (TSB) at 37°C for 24 h. For bio lm formation,

24-well plates containing 1 ml of TSB were inoculated

with 50 ml (1.5 x 108 bacteria/ml) of a bacterial suspen-

sion of each of the P. aeruginosa strains and incubated

at 37°C for 24 h. Bio lms were washed with phosphate

buffer solution (PBS) (pH 7.4) and  xed with 2% forma-

lin at 4°C overnight. Subsequently, the  xative solution

was removed with PBS and the  lms were stained with 1

ml of 1% crystal violet for 15 min. Excess crystal violet

was removed and 1 ml of methanol at 70% was added

for quanti cation of the bio lm to an optical density of

600 nm.All imipenem resistant isolates were tested for

MBL by Imipenem- EDTA combined disc test (CDT).

The Imipenem- EDTA combined disc test (CDT) was

performed as described by the method of Yong et al.

(2002). The test organisms were inoculated on the plates

with seeded Mueller Hinton agar as recommended by the

CLSI. A 0.5 M EDTA solution was prepared by dissolving

18.61g of EDTA in 100 ml of distilled water and adjust-

ing its pH 8.0. The Mixture was sterilized by autoclav-

ing. After two Imipenem (10µg) discs were placed on the

surface of agar plate, add  nally 4µl of 0.5 M concen-

tration of EDTA solution. Finally, the inhibition zones of

imipenem- EDTA discs were compared after 16 to 18 h

of incubation in air at 37°C. In the combined disc test,

if the increase in inhibition zone with imipenem- EDTA

disc was ≥7 mm it was considered as Metallo Beta Lac-

tamase positive (Lee et al. 2001).

The blood cancer causing P. aeruginosa was treated

with various different concentrations (0.2 mg/mL, 0.4

mg/mL, 0.6 mg/mL & 100g/mL) of ZrO

nanoparti-

cles no signi cant difference in the growth curve. The

growth experiment under the in uence of Zirconium

nanoparticles thus reveals the non-toxic nature of the

Zirconium nanoparticles in the bacterial system. In the

Rajasekar S., Vijayalakshmi S. and A. Mohankumar

382 ANTIBACTERIAL ACTIVITY OF ZRO

AGAINST METALLO BETA-LACTAMASE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

present study plasmid DNA was done by boiling prepa-

ration method (Holmes & Quigley, 1981; modi ed by

Riggs & McLachlan, 1986).

Antimicrobial activity of protease was carried out by

using well diffusion method. The Pseudomonas aerugi-

nosa was inoculated in LB broth and incubated for 24

hrs at 37

C. The turbidity of the broth was adjusted at

0.5 (optical density) using Spectrophotometer. The bacte-

rial culture was inoculated on MHA plates using sterilized

cotton swab. Allow it to dry for 2 – 5 min. Well was made

using sterile cork borer. Various different concentrations

(0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL & 100g/mL) of dif-

ferent µl of ZrO

nanoparticles (50µl, 100µl and 150µl)

was impregnated in to the well. The plates were swabbed

with Pseudomonas aeruginosa. The plates were examined

to measure the zone of inhibition around the well.

RESULTS AND DISCUSSION

The carcinogenic disease is caused by external factors,

such as tobacco, infectious organisms, and an unhealthy

diet, and internal factors, such as inherited genetic muta-

tions, hormones, and immune conditions. These factors

may act together or in sequence to cause cancer. Cur-

rently, emerging and reemerging infectious disease are

a major problem in public health and global econom-

ics. They are caused by different type of infection such

as drug resistant infection, mostly involving bacteria,

and many emerging pathogen is increasing signi cantly

over time because they are becoming progressively more

resistant to conventional antibiotic compounds. For

example, Pseudomonas aeruginosa has been reported as

an opportunistic pathogen and one of the most impor-

tant opportunistic bacteria, causing blood cancer by the

intrinsic resistance to many antimicrobial agents. So, the

need for the development of new antibiotics to counter

drug resistance in bacterial pathogens has been stressed

by various researchers worldwide.

Totally 100 carcinogenic blood samples were collected

from the jugular vein with a sterile syringe of different

age group of blood cancer patients in different clinics

and PHC (Primary Health Centre) from in and around

PLATE 1. Isolated of colonies Pseudomonas aeruginosa from Blood

Cancer

Rajasekar S., Vijayalakshmi S. and A. Mohankumar

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ANTIBACTERIAL ACTIVITY OF ZRO

AGAINST METALLO BETA-LACTAMASE 383

Tirupur and Erode District. 80 isolates of Pseudomonas

aeruginosa were isolated from the samples. The can-

cer causing leading pathogen Pseudomonas aeruginosa

strains were con rmed by comparing the results with

standard biochemical test and 16S-rDNA gene sequence.

In the study 28.75% of blood cancer causing Pseu-

domonas aeruginosa isolates was found to be MBL pro-

ducers. These isolates exhibited a ≥ 7 mm zone enhanced

for Imipenem and EDTA combined than the Imipenem

disks alone. All the MBL producing isolates were multi

drug resistant, most of which showed resistance to

more than twelve antibiotics out of the twenty anti-

biotics tested. Twenty three isolates: PARS01, PARS02

PARS03, PARS05, PARS06, PARS21, PARS41, PARS43,

PARS44, PARS47, PARS51, PARS56, PARS58, PARS60,

PARS61, PARS62, PARS63, PARS64, PARS74, PARS75,

PARS74, PARS75, PARS77, PARS78 and PARS80 which

was showed more than above 50% resistance used for

Metallo Beta Lactamase production by Imipenem (IMP)-

EDTA Combined Disk test of (Yong et al. 2002). The

maximum zone of inhibition (mm) produced by the

strain No. PARS21 and PARS 77 which showed 40 mm

followed by minimum amount of zone of inhibition

produced by the strain No. PARS56 and PARS64 which

showed 30mm (Plate: 8; Table: 16). Electrophoretic

analysis of the plasmid DNA prepared was carried out

by agarose gel electrophoresis on 0.7%. Nineteen car-

cinogenic isolates (PARS01, PARS02, PARS03, PARS05,

PARS06, PARS21, PARS41, PARS43, PARS44, PARS47,

PARS51, PARS56, PARS58, PARS60, PARS61, PARS63,

PARS64 and PARS80) which was showed more than

50% resistance against tested antibiotics were selected

for plasmid isolation.

The antibacterial activity of ZrO

nanoparticles was

studied by agar well diffusion method. Three differ-

ent concentrations (0.2%, 0.4% and 0.6%) were used

in this assay against leukemia (blood cancer) causing

Pseudomonas aeruginosa. The maximum zone of inhibi-

tion 18mm, 19mm and 22mm was observed in strain

no. PARS47 followed by the minimum zone of inhibi-

tion 15mm, 16mm and 17mm was observed in strain no.

PARS64 and PARS56 at 150l concentration of different

µg- 50, 100 and 150 of ZrO

. Mahrukh Khattak et al.,

(2013) determined the frequency of Pseudomonas aerug-

inosa in middle and outer ear and to check the antibiotic

susceptibility pattern of commonly used antibiotics. The

pathogen was highly sensitive to Gentamycin 30 (71.4%),

followed by Aztreonam 28(66.6%), Ce xime 22(52.3%),

Imipenem 18(42.8%), amikacin 15(35.5%) and Cip-

ro oxacin 12(28.5%). Females were more susceptible

to Pseudomonas infections. Isolates of Pseudomonas

PLATE 2. Detection of Metallo

-Lactamase by Imipenam EDTA

Combined Disc Test

PLATE 3. Bio lm production by carcinogenic patho-

gen Pseudomonas aeruginosa

PLATE 4. Isolation of plasmid from Pseudomonas aeruginosa

Rajasekar S., Vijayalakshmi S. and A. Mohankumar

384 ANTIBACTERIAL ACTIVITY OF ZRO

AGAINST METALLO BETA-LACTAMASE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

aeruginosa from samples showed highest sensitivity

(71.4%) to Gentamycin while maximum resistance was

showed to Cipro oxacin (47.6%). In this research, simi-

lar results were found in all the Pseudomonas aerugi-

nosa in blood cancer origin were more highly sensitive

to Imipenem (97.5%).

Resistance to imipenem has been found to be inde-

pendent of -lactamase production and in P. aeruginosa

has been attributed to diminished expression of certain

outer membrane proteins (Buscher et al., 2000). More

than 80% of isolates in this study were sensitive to imi-

penem (97.5%). Compared with results of a study con-

ducted at the Lagos University Teaching Hospital (LUTH)

in which 12.5% were resistant to imipenem, in this study

only 5.4% Pseudomonas strains were resistant. Imipe-

nem is a drug that is not readily available in our envi-

ronment and its cost is also prohibitive.

Plasmid analysis of the multi-resistant strains showed

that 18 of the Pseudomonas strains harbored plasmids,

eleven of which had similar plasmid band patterns of

1-7 plasmid bands having low to intermediate molecular

weights. Plasmid prevalence was higher in the strains

from blood cancer. Acquisition of mobile genetic ele-

ments is known to be the main mechanism for short term

accumulation of resistance determinants in bacterial

genomes (Liu et al., 2000).Our  nding that 18(78.75%)

out of the 46 Pseudomonas isolates contained plasmids

with seven different plasmid pro les, coincides with

that reported by Tsakris et al., (1992) who found that

10 isolates harbored plasmids out of 35 multi resistant

pseudomonas strains, with 6 different plasmid pro les.

In another study, Poh et al., (1988) detected 11 different

plasmid pro les in Ps. aeruginosa isolates.

Bio lm producing organisms are responsible for

many recalcitrant infections and are notoriously dif -

cult to eradicate. They exhibit resistance to antibiotics

by various methods like restricted penetration of anti-

biotic into bio lms, decreased growth rate and expres-

sion of resistance genes (Hachem et al., 2007). There are

various methods for bio lm detection (Poole, 2004). In

the present study the number of isolates showing strong

bio lm producers were 41(64%) and moderate bio lm

producers were 39(36%) by Tissue culture plate method

similar to the study done by Afreenish Hassen et al.,

(2011). In their study noted that out of 110 isolates from

different clinical samples tested for bio lm production,

the number of bio lm producers identi ed by Tissue cul-

ture plate method (TCP) was 70(64.7%) and non or weak

bio lm producers were 40(36.3%). The recent study has

performed the Tissue culture plate method by addition

of 1% glucose in trypticase soy broth. Addition of sugar

helps in bio lm formation16. This was also reported by

studies conducted by Mathur et al., (2006).

Metallo-beta-lactamase enzyme is an emerging threat

and cause of concern for physician. The metal ion active

sites appear to decrease their susceptibility to beta lac-

tamase inhibitors and enable them to hydrolyze broad

spectrum including carbapenems. The Metallobeta-lac-

tamase are plasmid mediated, so the resistance can be

spread among hospital pathogen and will cause prob-

lems in treating infections (Mehul Chaudhari et al.,

2011). In present study, attempt was made to detect

Metallobeta- lactamase producing Pseudomonas aer-

uginosa. Of 80 isolates of Pseudomonas aeruginosa, 23

(2.5%) were resistance to imipenem. All 23 isolates were

found to be MBL producers. Of 23 isolates of MBL, 23

(28.75%) were isolated from blood cancer patient. The

prevalence of detect Metallo-beta-lactamase producing

Pseudomonas aeruginosa in our setup if 2.5%.

Ae Mftah et al., (2015) studied the sulphated zirco-

nia nanoparticles showed high antimicrobial activity

against both Gram-positive and Gram-negative bac-

teria. It was found that the nanoparticles showed the

highest activity against Pseudomonas aeruginosa and

methicillin-resistant S. aureus, followed by Bacillus

subtilis and Salmonella choleraesuis. The  rst time that

the exposure of cancer cells to sulphated zirconia nano-

particles (3.9–1,000 g/mL for 24 hours) resulted in a

dose-dependent inhibition of cell growth, as determined

by (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide assays. Similar promising results were observed

for reducing bacteria functions. In this manner, this

study demonstrated that sulphated zirconia nanoparti-

cles with bronsted acidic sites should be further studied

for a wide range of anticancer and antibacterial appli-

cations. Followed by these present study similar results

of antimicrobial ability of ZrO2 at different concentra-

tion (0.2%, 0.4% and 0.6%) used against blood cancer

causing Pseudomonas aeruginosa. Zirconium oxide was

PLATE 5. Antibacterial activity of

ZrO

against Metallo beta-lactamase

and bio lm producing carcinogenic

Pseudomonas aeruginosa

Rajasekar S., Vijayalakshmi S. and A. Mohankumar

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ANTIBACTERIAL ACTIVITY OF ZRO

AGAINST METALLO BETA-LACTAMASE 385

used and it shows prominent anti carcinogenic activity

against test pathogen.

CONCLUSION

The study also highlights that ML incidence is increas-

ing in our region. The metallo beta-lactamase and bio lm

production is the most worrisome resistant mechanisms

observed in P. aeruginosa. Emergence of antimicrobial

resistance by pathogenic bacteria is a major health prob-

lem in recent years. The resistance may spread rapidly

to various species of Gram-negative bacilli; therefore, to

prevent the further spread of ML producers, it is essen-

tial to rapidly detect ML-positive isolates to aid infection

control. Recurrently, tiny nanoparticles, far smaller than

the width of a human hair, might help the body’s own

immune system  ght tumors. Moreover, it is reported here

that these novel metallo nanopar ticles (ZrO

) comprises

of well-known inhibitory and bactericidal effects against

cancer causing Pseudomonas aeruginosa.

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