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Diversity of Extended Spectrum β-lactamases among Multi Drug Resistant Clinical Isolates of Pseudomonas aeruginosa Collected fromTertiary Care Hospitals of Peshawar, Pakistan

PJZ_53_3_885-893

Diversity of Extended Spectrum β-lactamases among Multi Drug Resistant Clinical Isolates of Pseudomonas aeruginosa Collected fromTertiary Care Hospitals of Peshawar, Pakistan

Amjad Ali, Kafeel Ahmad*and Shaista Rahat

Centre of Biotechnology and Microbiology, University of Peshawar, Pakistan

ABSTRACT

Pseudomonas aeruginosa is an opportunistic pathogen and the leading cause of nosocomial infection worldwide. This bacterium produces enzymes known as extended spectrum β-lactamases which render broad spectrum cephalosporins and penicillins inactive. This study reports antibiotic susceptibility pattern, multiple antibiotic resistance (MAR) index and prevalence of extended spectrum β-lactamases among clinical isolates of P. aeruginosa collected from tertiary care hospitals of Peshawar, Pakistan. A total of 187 P. aeruginosa isolates were collected. Antibiotic susceptibility was evaluated by Kirby Bauer disc diffusion method using nineteen different antibiotics and multiple antibiotic resistance (MAR) index was determined. Prevalence of extended spectrum β-lactamases was studied by double disc synergy test. The ESBL genes blaCTX-M, blaOXA-10, blaPER-1, blaSHV and blaTEM were analyzed by PCR amplification among the isolates. Susceptibility to antibiotics was: imipenem (85.02%), meropenem (82.88%), cefepime (76.47%), piperacillin-tazobactam (76.47%), colistin (74.86%), ciprofloxacin (74.33%), piperacillin (72.19%), ceftazidime (68.98%), ofloxacin (68.44%), amikacin (66.84%), cefoperazone (66.31%), carbenicillin (66.31%), gentamicin (64.7%), tobramycin (64.7%), aztreonam (52.4%), ticarcillin (42.78%), ceftriaxone (32.08%), cefotaxime (15.5%), amoxicillin-clavulanic acid (6.41%). A total of 36.89% (n=69) isolates showed multi drug resistance. The MAR index of 34.22% (n=64) isolates was higher than 0.2. Phenotypic ESBL production was observed in 21.39% (n=40) isolates. Prevalence of blaOXA-10, blaCTX-M, blaTEM and blaSHV was 36.89% (n=69), 20.85% (n=39), 5.34% (n=10) and 3.2% (n=6) respectively. PER-1 gene was not detected. Resistance to antibiotics is increasing in P. aeruginosa which is a matter of concern and needs proper management. Non-selective and over use of antibiotics should be avoided and proper control measures should be taken to avoid the spread of these multi-drug resistant strains.


Article Information

Received 12 July 2019

Revised 30 September 2019

Accepted 28 January 2020

Available online 19 March 2021

Authors’ Contribution

AA conducted the experiments, compiled the data and wrote the manuscript. SR contributed towards manuscript writing and proof reading. KA designed the project, supervised the work, contributed towards data analysis, manuscript writing and proof reading.

Key words

Pseudomonas aeruginosa, MDR, MAR Index, ESBL, blaOXA-10, blaCTX-M, blaPER-1, blaTEM, blaSHV

DOI: https://dx.doi.org/10.17582/journal.pjz/20190712160705

* Corresponding author: kafeelpbg@gmail.com

0030-9923/2021/0003-0885 $ 9.00/0

Copyright 2021 Zoological Society of Pakistan



INTRODUCTION

Pseudomonas aeruginosa is one of the main causes of nosocomial infections like burn infections, wounds infections, urinary tract infections, pneumonia, bacteremia, otitis externa, endophthalmitis, meningitis and infections in cystic fibrosis patients (Branski et al., 2009). These infections could develop into more severe form in immune compromised patients like cancer and neutropenic patients (Bodey et al., 1983). The global emergence of multi-drug resistant P. aeruginosa is serious health issue as P. aeruginosa resistant to several classes of antibiotics such as penicillin, cephalosporin, aminoglycoside, quinolone and carbapenem have been reported (Dundar and Otkun, 2010). Antimicrobial resistance mechanisms in P. aeruginosa include multidrug efflux pumps, outer membrane impermeability to antibiotics, enzymatic degradation of antibiotics and target site modification (Lambert, 2002; Mesaros et al., 2007). Production of extended spectrum beta lactamases (ESBLs) by P. aeruginosa is an important mechanism to inactive antibiotics. These enzymes could hydrolyze penicillins, extended spectrum cephalosporins such as, ceftriaxone, ceftazidime, cefotaxime and the monobactam aztreonam (Paterson and Bonomo, 2005; Khanfar et al., 2009). However, they have no effect on cephamycins or carbapenems and their activity is inhibited by clavulanic acid (Paterson and Bonomo, 2005). The global spread of ESBL producing P. aeruginosa pose a serious health threat.

Extended spectrum β-lactamase producing bacteria were first reported in Germany in 1983 (Knothe et al., 1983). Different variants of ESBLs such as TEM, PER, SHV, GES, VEB and CTX-M have been reported in P. aeruginosa of different geographical origins (Aktas et al., 2005; Al Naiemi et al., 2006; Celenza et al., 2006; Zhao and Hu, 2010). Cefotaximase-Munich (CTX-M) beta lactamase was first isolated from Escherichia coli recovered from ear exudate of newly born baby in Munich, Germany (Bauernfeind et al., 1990). These β lactamases have been divided into five groups based on amino acid sequence i.e. CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25 (Paterson and Bonomo, 2005; Gupta, 2007). All these CTX-M β lactamases have been reported from different countries including Japan, Germany, Argentina, Poland, Taiwan, France, Spain, Brazil, China, Korea and Canada (Canton et al., 2012). These enzymes possess hydrolytic activity against cefotaxime and have approximately 40 % or less identity with other β-lactamases such as TEM and SHV (Paterson and Bonomo, 2005). Oxacillinases (OXA) are β-lactamases that could hydrolyze oxacillin and have been reported in P. aeruginosa (Naas et al., 2008; El-Shouny et al., 2018; Odumosu et al., 2016). The OXA type variants, OXA-10 and OXA-13, weakly hydrolyze cephalosporin (cefotaxime and ceftriaxone) and aztreonam (Naas et al., 2008). The Pseudomonas extended resistance (PER-1) β-lactamase of P. aeruginosa can efficiently hydrolyze third generation cephalosporins, penicillins and aztreonam but has no effect on cephamycins and carbapenems (Aktas et al., 2005; Nordmann and Naas, 1994; Opus et al., 2017; Qing et al., 2014). Sulfhydryl variable (SHV) type β-lactamases i.e. SHV-1 efficiently hydrolyze cefotaxime but slightly hydrolyze ceftazidime (Paterson and Bonomo, 2005). More than 50 SHV β-lactamases have been identified which are derived either from SHV-1 or SHV-2 (Paterson and Bonomo, 2005; Gupta, 2007; Peymani et al., 2017). In 1965, TEM-1 β-lactamase was first confirmed in Escherichia coli isolated from a patient named Temoneira in Athens (Datta and Kontomichalou, 1965). Such β-lactamases could hydrolyze β-lactam antibiotics such as penicillins and cephalosporins (Salverda et al., 2010; Hassuna et al., 2015). The spread of multiple antibiotic resistant P. aeruginosa in hospital environments has been reported across the world (Krumperman, 1983; Paul et al., 1997).

Infectious diseases are highly prevalent in Pakistan, however; there is scarcity of data on genotypic characteristics of locally prevalent bacterial pathogens. This study was aimed at investigating phenotypic and genotypic characterization of extended spectrum beta lactamases among clinical isolates of P. aeruginosa isolated from different clinical specimens in tertiary care hospitals of Peshawar, Khyber Pakhtunkhwa Pakistan.

MATERIALS AND METHODS

Bacterial isolates

A total of 187 P. aeruginosa isolates were collected from clinical specimens in tertiary care hospitals of Peshawar, Pakistan during 2014-2016. Among these, 74 isolates were recovered from pus, 34 from urine, 24 from sputum, 21 from wound, 12 from bronchial wash, 8 from cerebrospinal fluid, 6 from blood, 5 from high vaginal swab and 3 from diabetic foot. The cultures were grown on MacConkey agar (Oxoid, UK). Pure isolates were identified as P. aeruginosa using morphological and biochemical tests (Parija, 2006).

Antibiotic sensitivity

Antibiotic sensitivity was evaluated using Kirby Bauer disc diffusion method as suggested by the Clinical Laboratory Standard Institute (Clinical Laboratory Standard Institute, 2007; Clinical Laboratory Standard Institute, 2014). The antibiotics (Oxoid, UK) used were: Amoxicillin-clavulanic acid (30 µg), Cefotaxime (30 µg), Piperacillin-tazobactam (110 µg), Cefoperazone (75 µg), Ceftazidime (30 µg), Ceftriaxone (30 µg), Gentamicin (10 µg), Meropenem (10 µg), Cefepime (30 µg), Aztreonam (30 µg), Carbenicillin (100 µg), Imipenem (10 µg), Ticarcillin (75 µg), Piperacillin (100 µg), Amikacin (30 µg), Ciprofloxacin (5 µg), Tobramycin (10 µg), Ofloxacin (5 µg) and Colistin (10 µg). Bacterial colonies were suspended in normal saline and turbidity was adjusted by comparing with 0.5 McFarland standard. Bacterial suspension was inoculated on Muller Hinton agar (Oxoid, UK) plate and discs were placed on the medium at equal distances. The cultures were incubated at 37 ˚C for 18-24 hrs. and zones of inhibition were measured.

Determination of multiple antibiotic resistance index

Multiple antibiotic resistance index for each isolate of P. aeruginosa was determined using the formula, MAR index= a/b where ‘a’ represent the number of antibiotics to which isolate show resistant, where ‘b’ represent total number of antibiotics used (Krumperman, 1983; Sandhu et al., 2016).

Phenotypic detection of extended spectrum β-lactamases

Double disc synergy test was used for detection of ESBLs production (Jarlier et al., 1998). Bacterial lawn was made on Muller Hinton Agar and amoxicillin-clavulanic acid disc was applied in the center. Discs of aztreonam, cefepime, cefotaxime and ceftazidime were placed 15-20 mm away from the disc of amoxicillin-clavulanic acid. The plates were incubated at 37 ˚C for 18 h and inhibition zones were measured. Increase in size of inhibition zone around one or more cephalosporin discs and aztreonam towards amoxicillin-clavulanic acid disc showed presence of ESBL production.

DNA extraction

GeneJET Genomic DNA purification kit (Thermo Scientific, Lithuania, #K0721) was used for isolation of bacterial genomic DNA. Isolated DNA was preserved at -20˚C.

Molecular detection of ESBLs

Previously reported primers were used for the amplification of blaCTX-M, blaOXA-10, blaPER-1, blaSHV, and blaTEM genes (Farshadzadeh et al., 2014; Peerayeh et al., 2014). PCR reaction mix (25 µl) contained 12.5 µl SuperHot Master Mix (BIORON, Cat. No. 119102), 1 µl of each primer (0.5 µM), 1 µl genomic DNA and 9.5 µl molecular grade water (Sigma-Aldrich, US). Reaction conditions consisted of initial denaturation (95 ºC for 5 min), followed by 30 cycles of denaturation (94 ºC for 1 min), annealing (55 ºC for blaCTX-M and blaTEM; 57 ºC for blaOXA-10; 48 ºC for blaPER-1; 60 ºC for blaSHV) and extension (72 ºC for 1 min). Final extension was carried out at 72 ºC for 5 min. PCR products were analyzed using agarose gel (1.5 %) and 100 bp DNA ladder (BIORON, Cat. No. 304105) was used as size marker.

Table I. Antimicrobial susceptibility of Pseudomonas aeruginosa isolates.

S. No.

Antimicrobial

Susceptible, No. (%)

Intermediate, No. (%)

Resistant, No. (%)

1.

TZP

143 (76.47%)

26 (13.90%)

18 (9.62%)

2.

AMC

12 (6.41%)

20 (10.69%)

155 (82.88%)

3.

CTX

29 (15.50%)

80 (42.78%)

78 (41.71%)

4.

CAZ

129 (68.98%)

7 (3.74%)

51 (27.27%)

5.

CRO

60 (32.08%)

56 (29.94%)

71 (37.96%)

6.

CFP

124 (66.31%)

20 (10.69%)

43 (22.99%)

7.

FEP

143 (76.47%)

6 (3.20%)

38 (20.32%)

8.

ATM

98 (52.40%)

49 (26.20%)

40 (21.39%)

9.

IPM

159 (85.02%)

3 (1.60%)

25 (13.36%)

10.

MEM

155 (82.88%)

0 (0%)

32 (17.11%)

11.

TIC

80 (42.78%)

58 (31.01%)

49 (26.20%)

12.

PIP

135 (72.19%)

32 (17.11%)

20 (10.69%)

13.

CB

124 (66.31%)

12 (6.41%)

51 (27.27%)

14.

CN

121 (64.70%)

17 (9.09%)

49 (26.20%)

15.

AK

125 (66.84%)

16 (8.55%)

46 (24.59%)

16.

TOB

121 (64.70%)

14 (7.48%)

52 (27.80%)

17.

CIP

139 (74.33%)

7 (3.74%)

41 (21.92%)

18.

OFX

128 (68.44%)

11 (5.88%)

48 (25.66%)

19.

CT

140 (74.86%)

0 (0%)

47 (25.13%)

 

AMC, Amoxicillin-clavulanic acid (30 µg); CTX, Cefotaxime (30 µg), TZP, Piperacillin-tazobactam (110 µg); CFP, Cefoperazone (75 µg); CAZ, Ceftazidime (30 µg); CRO, Ceftriaxone (30 µg); CN, Gentamicin (10 µg); MEM, Meropenem (10 µg); FEP, Cefepime (30 µg); ATM, Aztreonam (30 µg); CB, Carbenicillin (100 µg); IPM, Imipenem (10 µg); TIC, Ticarcillin (75 µg); PIP, Piperacillin (100 µg); AK, Amikacin (30 µg); CIP, Ciprofloxacin (5 µg); TOB, Tobramycin (10 µg); OFX, Ofloxacin (5 µg) and CT, Colistin (10 µg).

RESULTS

Antibiotic sensitivity data is given in Table I. Resistance to cephalosporin third generation antimicrobials cefoperazone, ceftazidime, ceftriaxone and cefotaxime were 22.99%, 27.27%, 37.96% and 41.71%, respectively. Resistance to cephalosporin fourth generation cefepime was 20.32%. Resistance to pencillins antimicrobials i.e. piperacillin, ticarcillin and carbenicillin was 10.69%, 26.2% and 27.27%, respectively. Resistance to aminoglycoside antimicrobials amikacin, gentamicin and tobramycin was 24.59%, 26.2% and 27.8%, respectively. Resistance to fluoroquinolones antibiotics i.e. ciprofloxacin and ofloxacin was 21.92% and 25.66%, respectively. Resistance to carbapenem antimicrobials imipenem and meropenem was 13.36% and 17.11%, respectively. Resistance to monobactam antibiotic aztreonam was 21.39%. Resistance to polymyxin antibiotic colistin was 25.13%. All imipenem resistant isolates of P. aeruginosa were susceptible to colistin.

 

Table II. Prevalence of MDR isolates of Pseudomonas aeruginosa in different samples.

S. No

Specimen type

MDR P. aeruginosa

(n, %), (n = 69)

1.

Pus

30 (43.47)

2.

Urine

11 (15.94)

3.

Sputum

06 (8.69)

4.

Wound

10 (14.49)

5.

Bronchial wash

05 (7.24)

6.

Blood

03 (4.34)

7.

Cerebrospinal fluid

04 (5.79)

 

Table III. MAR index of Pseudomonas aeruginosa isolates (n=187).

MAR index

Number of isolates, (%)

0

27 (14.43)

0.05

41 (21.92)

0.10

34 (18.18)

0.2

21 (11.22)

0.3

14 (7.48)

0.4

13 (6.95)

0.5

5 (2.67)

0.6

10 (5.34)

0.7

11 (5.88)

0.8

5 (2.67)

0.9

6 (3.2)

 

A total of 36.89% (n=69) isolates showed multiple drug resistance (MDR) having resistance against three or more drug classes (Table II). Multiple antibiotic resistance (MAR) index values for isolates of P. aeruginosa are given in Table III and Table IV. In total, 34.22% (n=64) isolates showed MAR index greater than 0.2 and 54.54% (n=102) isolates showed MAR index value less than 0.2. Source wise MAR index of higher than 0.2 for the isolates was: blood (4.68% isolates), cerebrospinal fluid (6.25%), bronchial wash 7.81%), sputum (9.37%), wound (14.06%), urine (15.62%) and pus (42.18%) as shown in Table IV. Highest multiple antibiotic resistance index (MARI) of 0.9 was observed for six isolates that were resistant to all tested antibiotics except colistin.

 

Table IV. Distribution of Pseudomonas aeruginosa isolates based on MARI value > 0.2 among different clinical specimens.

Specimen type

Isolates with MAR Index > 0.2 (n= 64)

Percentage (%)

Pus

27

42.18

Urine

10

15.62

Wound

9

14.06

Sputum

6

9.37

Bronchial wash

5

7.81

Cerebrospinal fluid

4

6.25

Blood

3

4.68

 

Table V. Phenotypically ESBL positive strains of P. aeruginosa.

S. No.

Specimen

No of P. aeruginosa isolates (n =187)

ESBL positive P. aeruginosa isolates (n = 40), n (%),

1.

Pus

74

14 (35 %)

2.

Urine

34

10 (25 %)

3.

Sputum

24

4 (10 %)

4.

Wound

21

5 (12.5 %)

5.

Bronchial wash

12

3 (7.5 %)

6.

Blood

6

2 (5 %)

7.

Cerebrospinal fluid

8

2 (5 %)

8.

High vaginal swab

5

0 (0%)

9.

Diabetic foot

3

0 (0%)

 

Out of 187 isolates, 21.39 % (n=40) were ESBL positive phenotypically (Fig. 1). Frequency of ESBL positive isolates was 5%, 5%, 7.5%, 10%, 12.5%, 25% and 35% from cerebrospinal fluid, bronchial wash, sputum, wound, urine and pus samples respectively as given in Table V. Genotypically, blaOXA-10, blaCTX-M, blaTEM and blaSHV genes were detected in 36.89% (n=69), 20.85% (n=39), 5.34% and 3.2% (n=6) isolates respectively (Supplementary Table I, Fig. 2), however, blaPER-1 was not detected. Among phenotypically ESBL positive isolates (n=40), blaOXA-10, blaCTX-M, blaTEM and blaSHV were observed in 80% (n=32), 62.5% (n=25), 12.5% (n=5) and 7.5% (n=3) isolates respectively (Supplementary Table I). Among phenotypically ESBL negative isolates (n=147), blaOXA-10, blaCTX-M, blaTEM and blaSHV were observed in 25.17% (n=37), 9.52% (n=14), 3.4% (n=5) and 2% (n=3) isolates respectively (Supplementary Table I).


 

DISCUSSION

P. aeruginosa is rapidly developing resistance against the prevalent antibiotics. Previously, ESBLs producing P. aeruginosa has been reported from different geographical locations (Manchanda and Singh, 2003; Ghafourian et al., 2015). However, little data is available regarding ESBL producing P. aeruginosa prevailing in the region. Current study showed 21.39% prevalence of ESBL producing P. aeruginosa isolated from different clinical specimens. Isolates from pus had maximum frequency of ESBL production followed by urine, wound, sputum, bronchial wash, cerebro-spinal fluid and blood samples. These results are in harmony with the findings of Aggarwal et al. (2008) and Shaikh et al. (2015) who reported 20.27% and 25.13% ESBL frequency respectively among P. aeruginosa isolates from various clinical samples. Imipenem and meropenem are broad spectrum carbapenems commonly used effectively against extended spectrum β-lactamase


 

producing strains (Shaikh et al., 2015). These two antibiotics were found effective against most of the isolates in current study. Similar results have been reported previously (Shaikh et al., 2015; Alikhani et al., 2014). Resistance to both imipenem (13.36%) and meropenem (17.11%) was also observed as reported previously (Pathmanathan et al., 2009; Hong et al., 2015). In current study, 36.89% isolates showed multi drug resistance (MDR). Ullah et al. (2009) observed 29.24% MDR frequency among clinical isolates of P. aeruginosa from burn patients. Alikhani et al. (2014) observed 88.7% MDR frequency among P. aeruginosa isolates from west of Iran. In current study, ceftazidime was found to be the most effective (68.98% susceptibility) antibiotic among the third generation cephalosporines. (Shahid et al. (2003) reported 83.3% susceptibility to ceftazidime in samples from North India. A low level of resistance to the aminoglycosides amikacin, gentamicin and tobramycin was observed in the current study. In contrast, a study from Isfahan reported 60% resistance to gentamicin, 62% to tobramycin and 70% to amikacin (Golshani et al., 2012). Resistance to aminoglycoside is due to acquisition of plasmids which produce aminoglycoside modifying enzymes (Hancock, 1998). These enzymes modify the aminoglycosides antibiotics by various mechanisms such as acetylation, adenylation, and phosphorylation which decrease the uptake or reduce the ribosomal interaction of enzymatically modified drugs (Hancock, 1998). In addition, the chromosome of P. aeruginosa has an aminoglycoside resistant gene aphA that is activated by certain mutations (Hancock, 1998). Among fluoroquinolones, ciprofloxacin showed good activity (74.33% susceptibility) in this study. A study from France reported 68% susceptibility to ciprofloxacin (Cavallo et al., 2007). Resistance to ofloxacin and ciprofloxacin was 25.66% and 21.92% respectively in the current study. Golshani et al. (2012) reported high level of resistance to fluoroquinolones. Resistance to quinolone is because of mutations in regulatory gene mexR that regulates mexAB-oprM genes of efflux system and, hence, expression of efflux system genes is enhanced, and quinolone are extruded (Ziha-Zarifi et al., 1999). Among penicillins, piperacillin showed maximum activity (72.19% susceptibility). This finding is in close agreement to the report of Llanes et al. (2013) who investigated P. aeruginosa isolates from cystic fibrosis in France. P. aeruginosa acquires resistance to penicillins mostly due to alteration in penicillin binding protein (Srikumar et al., 1999).

In this study, 34.22% (n=64) isolates showed multiple antibiotic resistance (MAR) index value higher than 0.2. A higher MAR index value is an indication of over-use of antibiotics in a location that contributes to evolution of resistant bacteria (Krumperman, 1983; Paul et al., 1997). A study from Turkey reported a high percentage (51.92%) of clinical isolates of P. aeruginosa with MAR index higher of than 0.2 (Guvensen et al., 2017). Another study from India found that 39% P. aeruginosa collected from area with high use of antibiotics had MAR index of higher than 0.2 (Bhuvaneshwari, 2017). A high MAR index value (0.6 to 0.9) was observed for 22 isolates of P. aeruginosa recovered from clinical specimens and hospital environment in Nigeria (Chika et al., 2017). This high MAR index was suggested to be linked to the development of multi drug resistant isolates of P. aeruginosa because of intensive use of antibiotics in Nigeria (Chika et al., 2017).

According to current findings, prevalence of blaCTX-M was 20.85%. Prevalence of the gene was 10.7% among isolates of P. aeruginosa isolated from a

hospital in Makkah, Saudi Arabia (Ahmed et al., 2015) and 19.6% among isolates of P. aeruginosa collected from a Brazilian tertiary care hospital (Polotto et al., 2012). A high frequency of blaOXA-10 gene was observed in this study that is in agreement with previous reports (Weldhagen et al., 2003; Poirel et al., 2001; Neyestanaki et al., 2014). Low prevalence (5.34%) of blaTEM was observed in this study in contrast to previous reports from Iran (61%) and China (20.5%) (Neyestanaki et al., 2014; Chen et al., 2015). Low prevalence (3.2%) of blaSHV was observed in this study. Previous reports from Iran and India showed 36% and 1.78% prevalence of blaSHV respectively (Toupkanlou et al., 2015; Bharti et al., 2016). According to our knowledge; this is the first report on prevalence of blaOXA-10, blaCTX-M, blaTEM and blaSHV in clinical isolates of P. aeruginosa collected from regional hospitals.

CONCLUSIONS

In conclusion, the prevalence of ESBL producing MDR P. aeruginosa of clinical origin was confirmed in the region. These findings demand for controlled use of antibiotics and proper management strategies both at community level and in hospital environments to prevent the dissemination of these resistant bacteria.

ACKNOWLEDGMENT

The financial support provided by Higher Education Commission (HEC) Islamabad, Pakistan under the “Indigenous PhD Fellowships for 5000 scholar phase-II” is deeply acknowledged.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20190712160705

Statement of conflict of interest

The authors declare there is no conflict of interest.

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