extracellular enzymes and mycotoxins as virulence factors...

BIOSCIENCES BIOTECHNOLOGY RESEARCH ASIA, August 2014. Vol. 11(2), 479-490

* To whom all correspondence should be addressed.Mob.: +91-9486120820; Fax: 0091422 2591865;E-mail: [email protected]

Extracellular Enzymes and Mycotoxins as Virulence Factors in Fusarium and Aspergillus Keratitis

Kanesan Panneer Selvam1, Y. Randhir Babu Singh2, Coimbatore Subramanian Shobana2*, Nair Kalesh Karunakaran2,

Csaba Vágvölgyi3, László Kredics3, Raid Saleem Al-Baradie4 and Palanisamy Manikandan4-5

1Department of Microbiology, MR Government Arts College, Mannargudi - 614 001, India2Department of Microbiology, Dr. GR Damodaran College of Science, Coimbatore – 641 014, India.

3Faculty of Science and Informatics, Department of Microbiology, University of Szeged, Szeged, Hungary.4Department of Medical Laboratory, Applied Medical Sciences College, Majmaah University,

Kingdom of Saudi Arabia.5Department of Microbiology, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology,

Coimbatore - 641 014, Tamilnadu, India

doi: http://dx.doi.org/10.13005/bbra/1298

(Received: 09 May 2014; accepted: 06 June 2014)

The incidence of Fusarium and Aspergillus keratitis is on alarming rise with its burden increasing by the fact that they can produce an array of extra cellular enzymes and mycotoxins which act as virulence factors. In this context, 154 isolates of Fusarium spp. and Aspergillus spp. were screened for extracellular enzymes production. Further, representative isolates from the two genera were analyzed for mycotoxin production. Both genera were able to produce an array of extracellular enzymes. It was noted that protease and lipase are actively involved in Fusarium and Aspergillus keratitis. Aflatoxins were detected from Aspergillus spp., however no mycotoxins were detected from Fusarium spp. The study concludes that the extra cellular protease and lipase are cause of concern since it play important roles in pathogenesis of Fusarium and Aspergillus keratitis.

Key words: Fusarium, Aspergillus, Keratitis, Extracellular enzymes, Mycotoxins.

Eye diseases affecting the cornea are a major cause of blindness worldwide 1 next to cataract. In developing countries such as India, it has been recognized as a “silent epidemic” 2. Over the decades, there has been an increase in the percentage of infectious keratitis caused by filamentous fungi 3-6. Further, its burden is

worsened by the fact that fungi secrete several extracellular hydrolytic enzymes 7 with potential role as virulence factors 8-10, however, research on extracellular enzymes production as a virulence factor of Fusarium and Aspergillus isolated from ocular infection is very less. Gopinathan et al. 11, Zhu et al. 12, Burda & Fisher 13 and Dudley et al. 14 have reported that extracellular protease excreted from Fusarium and Aspergillus spp. isolated from corneal ulcer have been implicated in corneal matrix degradation. Mycotoxins are a group of secondary metabolites produced by various filamentous fungi and are involved in the infectious process of the fungi and may serve as virulence

480 SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)

factors 15-18. The present study was designed to evaluate the correlation between extracellular enzymes and mycotoxin production by Fusarium and Aspergillus with clinical characteristics of the mycotic keratitis patients so as to establish that extracellular enzymes and mycotoxin can act as virulence factors in mycotic keratitis.

MATERIALS AND METHODS

Patients All the patients studied in the present series were presented at Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore, India, between July 2010 and June 2012. Further, the clinical details of selected patients were collected and analyzed accordingly.Collection and processing of specimens Corneal scraping was done by an ophthalmologist with the aid of Kimura’s spatula (sterilized by flaming or wiping with 70% ethyl alcohol and drying), applying multiple, moderately firm, unidirectional strokes, under slit lamp illumination and directly inoculated on blood agar, chocolate agar and potato dextrose agar (PDA). The culture plates were incubated at 37ºC for 5 days except for PDA, which was incubated at 25ºC for 7 days. The identification of fungi was based on colony morphology and microscopic observation using lactophenol cotton blue wet mount. In vitro screening of extracellular enzymes among Fusarium and Aspergillus spp. In the present study seven different enzymes viz., protease 19 cellulase 20, keratinase 21, elastase 22, lipase 23, DNase 24 and a- amylase 25 was screened based on the methodologies described in the literature. Briefly, the conidial suspension of the test isolates was prepared to achieve a concentration of 105 conidia / mL and 50 µl of the spore suspension was inoculated on respective assay medium. The enzyme activity index was calculated for the test isolate by dividing the enzyme lysis diameter by the colony diameter as described by Blanco et al. 26.In vitro screening of aflatoxigenic Aspergillus spp. isolated from keratitis patients Screening of aflatoxigenic A. flavus was performed based on the method described by Saito and Machida 27. Briefly, the test isolate of Aspergillus spp. was inoculated at the center of the

PDA and incubated at 25°C for 3-4 days and the colonies were subsequently exposed to ammonia vapour. A change in the color of the colony reverse and formation of pink orange coloration upon exposing it to ammonium vapour was taken as a positive activity of aflatoxin.Determination of aflatoxins and T2 toxin from Fusarium and Aspergillus spp. Mycotoxin extract was prepared as described by Shih and Marth 28 and Dai et al. 29. The mold was grown on PDA slants for 7 days at 28°C. Spores were harvested by adding sterile distilled water to the slants. Precisely, 2ml of the spore suspension (with an optical density value of 0.5 at 550 nm) was inoculated into sterile glucose–salt medium (for aflatoxin) and T2-Toxin extraction medium (for T2-Toxin) and incubated at 25°C for 15 days. Later, the culture was filtered using Whatman No.1 filter paper and the filtrate was extracted with chloroform (2 volume of chloroform / volume of the filtrate). The extract was evaporated in a water bath at a temperature of 80°C and the residue thus obtained was dissolved in 0.2 ml of chloroform and mixed thoroughly. The extract thus obtained was subjected to Gas Chromatography–Mass Spectrometry (GC–MS) analysis for T2-Toxin. Similarly, presence of aflatoxins was determined using High Performance Liquid Chromatography (HPLC).

RESULTS

A total of 68 Fusarium spp. and 86 Aspergillus spp. (A. flavus; 63, A. fumigatus; 14, A. niger; 5, A. terreus; 3, and A. tamarii; 1) were evaluated for their extracellular enzyme activity and the representative results of each enzyme assay are shown in figure 1 (A-G). Among the isolates, Aspergillus spp. showed more protease (82.5% of 86), and a- amylase (88.3% of 86) activities than that of Fusarium spp. (protease; 70.5% of 68, a- amylase; 57.3% of 68). However, Fusarium spp. were comparatively active with cellulase and lipase than Aspergillus spp. (cellulase; 72% of 86 and lipase; 68.6% of 86). Interestingly, the rest of the enzymes viz., DNase, elastase and keratinase showed to have considerably lower activities in both Fusarium and Aspergillus spp. Among Aspergillus spp. the predominant species of the study, A. flavus were noted to have considerable

481SELVAM et al., Biosci., Biotech. Res. Asia, Vol. 11(2), 479-490 (2014)

Table 1. Percentage (%) of Fusarium and Aspergillus species/isolates with extracellular enzyme activities

Isolates Extracellular enzymes

Protease α- amylase Cellulase Lipase DNase Elastase Keratinase

A. flavus 53 57 45 44 21 18 21 (n = 63) (84.1%) (90.4%) (71.4%) (69.8%) (33.4%) (28.5%) (33.4%)A. fumigatus 10 10 11 8 3 3 6 (n = 14) (71.4%) (71.4%) (78.5%) (57.1%) (21.4%) (21.4%) (42.8%)A. niger 4 5 4 4 1 Nil Nil(n = 5) (80%) (100%) (80%) (80%) (20%) A. terreus 3 3 1 2 1 Nil Nil (n = 3) (100%) (100%) (33.4%) (66.7%) (33.4%) A. tamari 1 1 1 1 1 Nil Nil(n = 1) (100%) (100%) (100%) (100%) (100%) Fusarium 48 39 61 59 26 13 8 (n = 68) (70.5%) (57.3%) (89.7%) (86.7%) (38.2%) (19.1%) (11.7%)

Nil- No activity

activities of protease (84.1%, 53 of 63) and lipase (69.8%, 44 of 63) enzymes (table 1). Except two of the 154 isolates involved in this study, all the isolates had a minimum of two different enzyme activities. A total of 51 different sets/groups of enzyme combinations were identified to be secreted combining both the fungi and are given in table 2. A maximum activity was noted with respect to protease, α - amylase, cellulase, and lipase (14.2%; 22 of 154) combinations, followed by protease, α - amylase, cellulase, lipase, and DNase (9% of 154). Interestingly, though many of the Aspergillus species had extracellular protease activities compared to Fusarium spp., the latter was found to have a higher EAI (1.1) than Aspergillus (1.09), particularly from A. terreus. A. terreus also had a higher cellulase EAI of1.19. However, there were no comparatively significant differences existing in enzyme activity indices of a- amylase, DNase, and elastase amongst Fusarium and Aspergillus (table 3). Clinical data of a total of 63 representative patients suffering from Fusarium and Aspergillus keratitis were retrieved to ascertain the correlation of extracellular enzyme activity with clinical conditions of the patient. It was noted that elastase, keratinase and DNase activity have little influence on clinical character of fungal keratitis patient. On the other hand, protease and lipase activities were found to have strong associations with severity, healing etc., amongst infectious Fusarium and

Aspergillus keratitis cases. While protease activity was found to be associated with 71% (10 of 14 cases) of Fusarium keratitis cases who were earlier characterized to have a ‘severe’ infection, lipase activity was noted in all the cases. The observations were noted to be more similar among Aspergillus keratitis also. The detailed analyses of extracellular enzyme activities and the recorded clinical features are provided in tables 4 and 5 for Fusarium and Aspergillus keratitis, respectively. In total, 124 Aspergillus spp. (82 A. flavus, 11 A. niger, 24 A. fumigatus, 5 A. terreus and 2 A. tamari) were screened for aflatoxin producing ability, of which 41 (33%) isolates were detected with aflatoxin activity and were categorized to be aflatoxigenic (figure 1 H&I). Majority of these isolates were identified to be A. flavus (39, 95.1% of 41) and 1 isolate each of A. fumigatus and A. terreus. Further, 4 isolates of A. flavus, (2 each positive and negative aflatoxigenic A. flavus) were specifically identified for B1, B2, G1 and G2 toxins using HPLC and the overall findings are presented in table 6. Of the two non-aflatoxigenic strains, one (Cu34/11) was confirmed to produce aflatoxin B1, B2, G1 and G2 using HPLC. Remarkably, the A. flavus strain Cu670/10 was detected with higher concentrations (1.096 µg/ml) of G1 mycotoxin and the HPLC peaks indicating different types of aflatoxin identified are shown in figure 2. Similarly, among fusaria, four representative isolates of F. solani were screened for T2 mycotoxin production using GC-MS analysis. However, no isolate was


Table 2. Percentage distribution of hydrolytic enzyme activities among Fusarium and Aspergillus spp

Enzyme/s Aspergillus Fusarium Total (n=86) (n=68) (%)

α - Amylase 1 - 1 (0.65%)Lipase - 1 1 (0.65%)Keratinase 1 1 2 (1.3%)Protease, α - amylase 3 - 3 (2%)Protease, cellulase - 1 1 (0.65%)Protease, Keratinase - 1 1 (0.65%)Protease, α - amylase, cellulase 5 1 6 (3.9%)Protease, α - amylase, lipase 3 2 5 (3.2%)Protease, lipase, elastase, keratinase 1 - 1(0.65%)Protease, α - amylase, cellulase, lipase 12 10 22 (14.2%)Protease, α - amylase, lipase, DNase 3 - 3(2%)Protease, α - amylase, cellulase, elastase 1 - 1(0.65%)Protease, α - amylase, cellulase, DNase 3 - 3(2%)Protease, α - amylase, cellulase, lipase, DNase 7 7 14 (9%)Protease, α - amylase, cellulase, lipase, elastase 1 3 4 (2.6%)Protease, α - amylase, cellulase, DNase,keratinase 1 - 1(0.65%)Protease, α - amylase, cellulase, lipase, keratinase 4 1 5(3.2%)Protease, α - amylase, cellulase, lipase, DNase, elastase 2 3 5(3.2%)Protease, α - amylase, cellulase, lipase, DNase, keratinase 3 - 3(2%)Protease, α - amylase, cellulase, lipase, elastase, keratinase 2 1 3(2%)Protease, α - amylase, cellulase, lipase, DNase, elastase, keratinase - 1 1(0.65%)Protease, α - amylase, cellulase, keratinase 1 - 1(0.65%)Protease, α - amylase, cellulase, elastase 1 - 1(0.65%)Protease, α - amylase, cellulase, elastase, keratinase 1 - 1(0.65%)Protease, α - amylase, cellulase, DNase, elastase 1 - 1(0.65%)Protease, α - amylase, lipase, elastase 1 - 1(0.65%)Protease, α - amylase, lipase, keratinase 3 - 3(2%)Protease, α - amylase, lipase, elastase, keratinase 2 - 2(1.3%)Protease, α - amylase, keratinase 3 - 3(2%)Protease, cellulase, lipase 2 9 11 (7.1%)Protease, cellulase, lipase, DNase - 3 3(2%)Protease, cellulase, lipase, DNase, elastase - 1 1(0.65%)Protease, cellulase, lipase, DNase, keratinase 1 1 2(1.3%)Protease, cellulase, keratinase 2 - 2(1.3%)Protease, cellulase, DNase, 1 2 3(2%)Protease, lipase, DNase 1 - 1(0.65%)α - Amylase, lipase, DNase, elastase 1 - 1(0.65%)α - Amylase, cellulase, lipase 4 4 8 (5.2%)α - Amylase, lipase - 1 1(0.65%)α - Amylase, cellulose, lipase, DNase 1 2 3(2%)α - Amylase, cellulose, lipase, elastase - 1 1(0.65%)α - Amylase, cellulase, lipase, keratinase 1 1 2(1.3%)α - Amylase, cellulase, elastase 1 - 1(0.65%)α - Amylase, cellulase - 1 1(0.65%)α - Amylase, cellulase, DNase 1 - 1(0.65%)α - Amylase, cellulase, lipase, DNase, elastase - 1 1(0.65%)α - Amylase, cellulase, lipase, DNase, keratinase 1 - 1(0.65%)α - Amylase, cellulase, lipase, elastase, keratinase 1 - 1(0.65%)α - Amylase, elastase 1 - 1(0.65%)Cellulase, lipase 1 4 5(3.2%)Cellulase, lipase, DNase, elastase - 1 1(0.65%)Cellulase, lipase, elastase - 1 1(0.65%)Cellulase, lipase, keratinase - 1 1(0.65%)DNase, lipase - 1 1(0.65%)Total 86 68 154 (100%)


Table 3. Mean extracellular enzymes activity indices (EAI) of Fusarium and Aspergillus spp.

Fungi Mean enzyme activity index

Protease α-amylase Cellulase Lipase DNase Elastase

A. flavus 1.04 1.1 1.09 1.19 1.1 1.2A. fumigatus 1.06 1.1 1.1 1.17 1.1 1.3A. niger 1.07 1.1 1.07 1.1 NA NAA. terreus 1.09 1.1 1.19 1.19 NA NAA. tamarii NA NA NA NA NA NAFusarium 1.1 1.1 1.13 1.2 1.08 1.2

NA – Not applicable

Fig. 1. Extracellular enzyme activity of Fusarium and Aspergillus and aflatoxin screening of Aspergillus spp.α-amylase (a), cellulase (b), elastase (c), DNase (d), lipase (e) and protease (f) indicated with formation of clear hallow zone around the fungal colonies. Keratinase activity (g) was indicated by diffusion of keratine azure in basal media. Aflatoxigenic positive (h) and negative (i) A. flavus based on ammonium vapour method. The arrows in horizontal and vertical directions indicates zone of enzyme hydrolysis and zone of fungal colonies respectively


Tab

le 4

. Per

cent

age

dist

ribu

tion

of

posi

tive

cor

rela

tion

bet

wee

n re

cord

ed c

lini

cal f

eatu

res

and

the

extr

acel

lula

r en

zym

e ac

tivi

ty a

mon

g F

usar

ium

ker

atit

is p

atie

nts

Cli

nica

l fea

ture

s E

xtra

cell

ular

enz

ymes

act

ivit

ies

(%)

P

rote

ase

Lip

ase

Ela

stas

e K

erat

inas

e C

ellu

lase

D

Nas

e α

-Am

ylas

e

Sev

erit

y

M

ild

(n =

6)

3 (5

0.0)

4

(66.

7)

1 (1

6.7)

4

(66.

7)

3 (5

0.0)

-

3 (5

0.0)

Mod

erat

e (n

= 1

1)

7 (6

3.6)

9

(81.

8)

2 (1

8.1)

2

(18.

1)

11 (

100)

3

(27.

2)

9 (8

1.8)

Seve

rity

(n

= 1

4)

10 (

71.4

) 14

(10

0)

4 (2

8.5)

-

10 (

71.4

) 6

(42.

8)

6 (4

2.8)

Fin

al o

utco

me

Hea

led

(n =

26)

18 (

69.2

) 22

(84

.6)

5 (1

9.2)

5

(19.

2)

19 (

73.0

) 7

(26.

9)

15 (

57.6

)N

ot h

eale

d (n

= 3

) 2

(66.

7)

3 (1

00)

1 (3

3.4)

1

(33.

4)

3 (1

00)

1 (3

3.4)

1

(33.

4)N

o fo

llow

up(

n =

2)

- 2

(100

) 1

(50.

0)

- 2

(100

) -

1 (5

0.0)

Surg

ical

inte

rven

tion

Req

uire

d (n

= 6

) 3

(50.

0)

6 (1

00)

1 (1

6.7)

-

3 (5

0.0)

2

(33.

4)

3 (5

0.0)

Not

req

uire

d (n

= 2

5)

17 (

68.0

) 21

(84

.0)

6 (2

4.0)

6

(24.

0)

21 (

84.0

) 7

(28.

0)

15 (

60.0

)D

ata

not a

vail

able

-

- -

- -

- -

Sym

ptom

sP

ain

and

redn

ess

#(n

= 1

4)

6 (4

2.8)

10

(71

.4)

2 (1

4.2)

4

(28.

5)

10 (

71.4

) 4

(28.

5)

8 (5

7.1)

Oth

ers

* (n

= 1

7)

14 (

82.3

) 17

(10

0)

5 (2

9.4)

2

(11.

7)

14 (

82.3

) 5

(29.

4)

10 (

71.4

)D

ata

not a

vail

able

-

- -

- -

- -

Per

iod

requ

ired

for

reco

very

<1

mon

th (

n =

11)

5

(45.

4)

9 (8

1.8)

2

(18.

1)

5 (4

5.4)

8

(72.

7)

2 (1

8.1)

6

(54.

5)>

1mon

th –

4 m

onth

s(n

= 1

1)

10 (

90.9

) 9

(81.

8)

1 (9

.0)

- 9

(81.

8)

5 (4

5.4)

7

(63.

6)>

5 m

onth

s (n

= 6

) 4

(66.

7)

6 (1

00)

2 (3

3.4)

1

(16.

7)

4 (6

6.7)

-

4 (6

6.7)

Dat

a no

t ava

ilab

le(n

= 3

) 1

(33.

4)

3 (1

00)

2 (6

6.7)

-

3 (1

00)

2 (6

6.7)

1

(33.

4)

# -

Sin

ce p

ain

and

redn

ess

was

the

mos

t com

mon

sym

ptom

, it w

as p

rior

itiz

ed a

gain

st th

e re

st.

* -

Sym

ptom

s in

clud

ed w

ater

ing,

pho

toph

obia

, irr

itat

ion,

itch

ing,

dis

char

ge, s

wel

ling

, def

ecti

ve v

isio

n et

c.


Tab

le 5

. Per

cent

age

dist

ribu

tion

of

posi

tive

cor

rela

tion

bet

wee

n re

cord

ed c

lini

cal f

eatu

res

and

the

extr

acel

lula

r en

zym

e ac

tivi

ty a

mon

g A

sper

gill

us k

erat

itis

pat

ient

s

Cli

nica

l fea

ture

s E

xtra

cell

ular

enz

ymes

act

ivit

ies

(%)

P

rote

ase

Lip

ase

Ela

stas

e K

erat

inas

e C

ellu

lase

D

Nas

e α

-Am

ylas

e

Sev

erit

y

M

ild

(n =

1)

1 -

- -

1 -

1M

oder

ate

(n =

14)

12

(85

.7)

6 (4

2.8)

2

(14.

2)

4 (2

8.5)

8

(57.

1)

3 (2

1.4)

13

(92

.8)

Seve

rity

(n

= 1

7)

16 (

94.1

) 12

(70

.5)

3 (1

7.6)

2

(11.

7)

15 (

88.2

) 4

(23.

5)

16 (

94.1

)F

inal

out

com

eH

eale

d (n

=23

) 23

(10

0)

11 (

47.8

) 2

(8.6

) 4

(17.

3)

18 (

78.2

) 6

(26.

0)

22 (

95.6

)N

ot h

eale

d -

- -

- -

- -

No

foll

ow u

p(n

= 9

) 6

(66.

7)

7 (7

7.8)

3

(33.

4)

2 (2

2.3)

6

(66.

7)

1 (1

1.2)

8

(88.

9)Su

rgic

al in

terv

enti

onR

equi

red

(n =

12)

12

(10

0)

7 (5

8.4)

2

(16.

7)

2 (1

6.7)

9

(75.

0)

3 (2

5.0)

12

(10

0)N

ot r

equi

red

(n =

20)

17

(85

.0)

11 (

55.0

) 3

(15.

0)

4 (2

0.0)

15

(75

.0)

4 (2

0.0)

18

(90

.0)

Dat

a no

t ava

ilab

le

- -

- -

- -

-Sy

mpt

oms

Pai

n an

d re

dnes

s #(

n =

13)

12

(92

.3)

7 (5

3.8)

1

(7.6

) 1(

7.6)

11

(84

.6)

1(7.

6)

13 (

100)

Oth

ers

* (n

= 1

9)

17 (

89.4

) 11

(57

.8)

4 (2

1.0)

5

(26.

3)

13 (

68.4

) 6

(31.

5)

17 (

89.4

)D

ata

not a

vail

able

-

- -

- -

- -

Per

iod

requ

ired

for

reco

very

<1

mon

th (

n =

5)

5 (1

00)

2 (4

0.0)

-

1 (2

0.0)

4

(80.

0)

1 (2

0.0)

4

(80.

0)>

1mon

th –

4 m

onth

s(n

= 1

0)

10 (

100)

4

(40.

0)

2 (2

0.0)

1

(10.

0)

7 (7

0.0)

3

(30.

0)

10 (

100)

> 5

mon

ths

(n =

9)

9 (1

00)

6 (6

6.7)

1

(11.

2)

2 (2

2.3)

7

(77.

8)

2 (2

2.3)

9

(100

)D

ata

not a

vail

able

(n =

8)

5 (6

2.5)

6

(75.

0)

2 (2

5.0)

2

(25.

0)

6 (7

5.0)

1

(12.

5)

7 (8

7.5)


detected with mycotoxin activities, interestingly the compounds such as vitamins (Cu482/11), sterols

(Cu598/11) and a carotenoid such as rhodopin were detected from them.

Table 6. Screening of aflatoxins based on preliminary (ammonium vapor method)

and HPLC methods among Aspergillus spp. isolated from corneal ulcer

S. Isolates(Strain) Ammonium vapour HPLC

No. technique Aflatoxin Result Concentration(µg/ml)

1. A. flavus(Cu34/11) Negative B1 Positive 0.047 B2 Positive 0.012 G1 Positive 0.002 G2 Positive 1.4702. A. flavus(Cu1149/10) Negative B1 Negative NA B2 Negative NA G1 Negative NA G2 Negative NA3. A. flavus(Cu670/10) Positive B1 Negative NA B2 Positive 0.802 G1 Positive 1.096 G2 Positive 0.5444. A. flavus(Cu832/10) Positive B1 Negative NA B2 Negative NA G1 Negative NA G2 Negative NA

NA – Not applicable

DISCUSSION

In pursuit to find the possible role of the extracellular enzymes of Fusarium and Aspergillus as a virulence factor in causing keratitis, it was observed that both the species of Fusarium and Aspergillus were able to produce an array of

extracellular enzymes and the production spectrum differed between them. It was also noted that enzymes (cellulase and α- amylase) related to plant pathogenesis were produced by both Fusarium (89.7% in α- amylase and 57.3% in cellulase) and Aspergillus spp. (88.3% in α- amylase and 72% in cellulase). The observation implied that Fusarium

Fig. 2. Production of aflatoxins (B1, B2, G1 and G2) by A. flavus (Cu34/11) as shown by HPLC chromatogram


and Aspergillus isolated from corneal ulcer could have chiefly originated from plant materials. F. solani isolated from mycotic keratitis patients and plants had demonstrated the same pathogenicity in young bean, corn, and tomato plants and also invoked an eye reaction (erythema) in rabbit eye tested under laboratory conditions30. Ajayi et al. 31 reported amylase production by Fusarium spp. and Mishra and Dadhich 32 determined higher activity of amylase and xylanase in A. niger than Fusarium spp. The rate of cellulase activity as recorded in the present study was found to be in agreement with a previous study 33, although Kwon et al. 34 observed no cellulase activity in Fusarium spp. Additionally, the present study found Fusarium and Aspergillus had lesser activities of DNase, elastase and keratinase compared to protease and lipase but presumably with no active role in causing and keratinase. The detection of elastase and keratinase activity has been documented from clinical fungi isolated from invasive aspergillosis 22, 26, 35. Although the study detected no keratinase activity from A. niger, A. terreus and A. tamarii, previous studies 36-37 have reported higher activity of these enzymes in these species compared to Fusarium spp. More importantly, Rhodes et al. 38 stated that all clinical isolates involved in invasive aspergillosis produced elastase, but not all isolates having elastase activity were associated with invasive disease. Alp and Arikan 35 reported the extracellular elastase, acid proteinase and phospholipase activities of clinical isolates of A. fumigatus, A. flavus and A. niger and suggested that the existence and degree of these enzyme activities in clinical Aspergillus strains tend to show species and strain based variations and potentially significant correlation between the existence of high phospholipase activity and development of invasive disease. The fact that Fusarium (89.7% of 68) had higher activity of lipase than that of Aspergillus (68.8% of 86) in the present study was more or less congruent with the available literature39-42. In the present study, protease activity was profoundly seen in Aspergillus spp. (83% of 86), especially in A. flavus (84% of 63) compared to Fusarium spp. (71% of 68). The finding is a cause of concern among ophthalmologists since protease was proven to be associated with cornea that showed progressive ulcer along with the

presence of dense inflammatory cells 11. Further, the pathogenic role of protease in microbial keratitis has been documented by various authors 12, 43. The strong reports on the virulent nature of protease suggested that protease activity was strongly associated with severe Fusarium and Aspergillus keratitis. It was further found that all the patients of Aspergillus caused keratitis and 50% of the Fusarium keratitis cases with a positive protease activity had to undertake surgical intervention. The Fusarium isolates from the all patients with severe infection, who further required surgical intervention and longer period of healing showed a positive lipase activity. Fliesler 44 commented that lipids and lipid soluble compounds are essential constituents of the cells and tissues that comprise the eye and defect in their synthesis, intracellular and extracellular transport and turnover underlie a variety of significant, common and often severely debilitating eye diseases. These probably answer the critical role of lipase produced by the pathogenic isolates of fusaria and aspergilli of the present study towards the overall increase in the severity of keratitis. The toxic effects of aflatoxins in the cornea involve haziness of the cornea and separation of corneal lamellae, in addition to infiltration by polymorphonuclear leucocytes 45. Leema et al. 46 reported aflatoxin production from A. flavus of which 80% was found to be aflatoxin B1. However, the present study observed aflatoxin B2, G1 and G2 being more common compared to aflatoxin B1. It was emphasized that if an A. flavus strain isolated from a human lesion is found to possess aflatoxin-producing ability, it may be necessary to treat the lesion with not only standard antifungals, but also with molecules that suppress the production, or neutralize the deleterious effects of aflatoxins 46. Although, the present study could not detect any mycotoxin from the species of Fusarium a study47 reported as much as 76% of Fusarium spp. including F. solani, F. oxysporum, F. incarnatum, F. dimerum, F. verticilloides, and F. chlamydosoprum producing fusaric acid, moniliformin or fumonisin B1. Similarly, other study 48 reported detection of T-2 Toxin, nivalenol, diacetoxyacirpenol and deoxynevalenol from Fusarium keratitis using GC-MS. Interestingly, the authors had also concluded that in vitro toxin production does not influence severity of the


infection. Conversely, stated Fusarium infections of the eye after traumatic inoculation were more severe when trichothecenes produced 47. The high frequency of aflatoxin production by the clinical A. flavus compared to environmental strain possibly represented a response to pressures (antifungal chemotherapy, toxic factors released from corneal epithelial cells, or infiltrating inflammatory cells) that are not conducive to an ideal existence 46. However, it was observed that mycotoxin profiles of clinical and environmental A. flavus and A. fumigatus isolates were homogenous, except for gliotoxin production, which was detected only in the group of clinical isolates of A. fumigatus 49. The reports of mycotoxin being detected from various species of Fusarium keratitis isolates 47-48 and the contradiction with the present study could be due to non-expression of mycotoxin producing genes. However, the study points out the fact that fusaria were able to implicate with keratitis in high frequency, thus confirming the aggressive nature of Fusarium spp. causing mycotic keratitis. Though the screening of aflatoxigenic Aspergillus using ammonium vapour method as in the present study was not highly reliable and confirmative (as only 50% of the isolates was re-confirmed by HPLC, this approach could still provide provisional information on the virulent nature of the isolates. Screening of aflatoxigenic Aspergillus based on emission of bright blue or blue-green fluorescent area around the colonies has been reported for testing mycotoxin production 50. Similarly, different other approaches have also been reported for screening afltoxigenic Aspergillus 27,

51-53. The GC-MS based detection of compounds such as vitamin, sterol from Fusarium spp. in this study had been already reported 48, 54. Nevertheless, in vitro sterol production was not related to the severity of the infection 48.

CONCLUSION

The burden of Fusarium and Aspergillus tends to be increasing with the fact that they are able to produce various extra cellular enzymes which play important roles in the pathogenesis of the disease. Protease and particularly lipase are actively involved in severe mycotic keratitis which warrants further investigation.

ACKNOWLEDGEMENTS

The authors of this study were supported by the Indian National Science Academy and the Hungarian Academy of Sciences within the frames of the Indo – Hungarian bilateral exchange program no. IA/INSA-HAS Project 2013-2015/189. The research of Cs.V. was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4.A/2-11-1-2012-0001 'National Excellence Program'.

REFERENCES

1. Whitcher, J.P., Srinivasan, M., Upadhyay, M.P. Corneal blindness: a global perspective. Bull World Health Org., 2001; 79: 214-221.

2. Whitcher, J.P., Srinivasan, M. Corneal ulceration in the developing world-a silent epidemic. Br. J. Ophthalmol., 1997; 81: 622-623.

3. Rosa, R.H., Jr Miller, D., Alfonso, E.C. The changing spectrum of fungal keratitis in South Florida. Ophthalmology., 1994; 101:1005-1013.

4. Xie, L., Dong, X., Shi, W. Treatment of fungal keratitis by penetrating keratoplasty. Br. J. Ophthalmol., 2001; 85:1070-1074.

5. Manikandan, P., Varga, J., Kocsubé, S., Anita, R., Revathi, R., Németh, T.M., Narendran, V., Vágvölgyi, C., Panneer S.K., Shobana, C.S., Babu Singh, Y.R., Kredics, L. Epidemiology of Aspergillus keratitis at a tertiary care eye hospital in South India and antifungal susceptibilities of the causative agents. Mycoses., 2012; 56: 26-33.

6. Bharathi, M.J., Ramakrishnan, R., Vasu, S., Meenakshi, R., Palaniappan, R. Aetiological diagnosis of microbial keratitis in South India - A study of 1618 cases. Indian J. Med. Microbiol., 2002; 20: 19-24.

7. Khan, M.S.A., Ahmad, I., Aqil, F., Owais, M., Shahid, M., Musarrat, J. Virulence and pathogenicity of fungal pathogens with special reference to Candida albicans, Combating Fungal Infections: Problems and Remedy. Springer-Verlag Berlin Heidelberg., 2010; 21-45.

8. Latge, J.P. Aspergillus fumigatus and aspergillosis. Clin. Microbiol. Rev., 1999; 12: 310-350.

9. Bouchara, J.P., Tronchin, G., Larcher, G., Chabasse, D. The search for virulence determinants in Aspergillus fumigatus. Trends Microbiol., 1995; 3: 327-330.

10. Tomee, J.F., Kauffman, H.F. Putative virulence factors of Aspergillus fumigatus. Clin. Exp. Allergy., 2000; 30: 476-484.

11. Gopinathan, U., Ramakrishna, T., Willcox, M.,


Rao, C.M., Balasubramanian, D., Kulkarni, A., Vemuganti, G.K., Rao, G.N. Enzymatic, clinical and histologic evaluation of corneal tissues in experimental fungal keratitis in rabbits. Exp. Eye Res., 2001; 72: 433-442.

12. Zhu, W.S., Wojdyla, K., Donlon, K., Thomas, P.A., Eberle, H.I. Extracellular proteases of Aspergillus flavus. Diagn. Microbiol. Infect. Dis., 1990; 13: 491-497.

13. Burda, C.D., Fisher, E.Jr. Corneal destruction by extracts of Cephalosporium mycelium. Am. J. Ophthalmol., 1960; 50: 926-36.

14. Dudley, M.A., Chick, E.W., Morgantown, W.A. Corneal lesions produced in rabbits by an extract of Fusarium moniliformae. Arch. Ophthalmol., 1964; 72: 346-50.

15. Keller, N.P., Hohn, T.M. Metabolic pathway gene cluster in filamentous fungi. Fungal Genet. Biol., 1997; 21: 17-29.

16. Desjardins, A.E., Proctor, R.H., Bai, G., McCormick, S.P., Shaner, G., Buechley, G., Hohn, T.M. Reduced virulence of trichothecene – nonproducing mutants of Gibberela zeae in wheat field tests. Mol. Plant – Microbe. Interact., 1996; 9: 775-781.

17. Iwata, K. Toxins produced by Candida albicans. Contr. Microbiol. Immunol., 1977; 4: 77-85.

18. Trapp, S.C., Hohn, T.M., McCormick, S., Jarvis, B.B. Characterization of the gene cluster for biosynthesis of macrocyclic trichothecenes in Myrothecium roridum. Mol. Gen. Genet., 1998; 257: 421-432.

19. Sharma, J., Singh, A., Kumar, R., Mittal, A. Partial purification of an alkaline protease from a new strain of Aspergillus oryzae AW T20 and its enhanced stabilization in entrapped Ca – Alginate beads. The Internet. J. Microbiol., 2006; 2: 2

20. Gautam, S.P., Bundela, P.S., Pandey, A.K., Jamaluddin, Awasthi, M.K., Sarsaiya, S. Optimization of the medium for the production of cellulase by the Trichoderma viride using submerged fermentation. Int. J. Environ. Sci., 2010; 1(4): 656-665.

21. Scott, J.A., Untereiner, W.A. Determination of keratin degradation by fungi using keratin azure. Med. Mycol., 2004; 42: 239-246.

22. Kothary, M.H., Chase, T., MacMillan, J.D. Correlation of elastase production by some strains of Aspergillus fumigatus with ability to cause pulmonary invasive aspergillosis in mice. Infect. Immun., 1984; 43:320-325.

23. Nara, G., Polloni, A.E., Remonatto, D., Arbter, F., Vardanega, R., Cechet, J.L., Luccio, M.D., de Oliveira, D., Treichel, H., Cansian, R.L., Rigo, E., Ninow, J.L. Isolation and screening of lipase-producing fungi with hydrolytic activity. Food

Bioproc. Technol., 2011; 4: 578-586.24. Sanchez, M., Colom, F. Extracellular DNase

activity of Cryptococcus neoformans and Cryptococcus gatti. Rev. Iberoam. Micol., 2010; 27: 10-13.

25. Prabakaran, M., Thennarasu, V., Ayeswariya, M.R., Bharathidasan, R., Chandrakala, N., Mohan, N. Comparative studies on the enzyme activities of wild and mutant fungal strains isolated from sugarcane field. Ind. J. Sci. Technol., 2009; 2: 46-49.

26. Blanco, J.L., Hontecillas, R., Bouza, E., Blanco, I., Pelaez, T., Muñoz, P., Molina, J.P., Garcia, M.E. Correlation between the elastase activity index and invasiveness of clinical isolates of Aspergillus fumigatus. J. Clin. Microbiol., 2002; 40:1811-1813.

27. Saito, M., Machida, S. A rapid identification method for aflatoxin-producing strains of Aspergillus flavus and A. parasiticus by ammonia vapor. Mycoscience., 1999; 40: 205-208.

28. Shih, C.N., and Marth, E.H. Some cultural conditions that control biosynthesis of lipid and aflatoxin by Aspergillus parasiticus. Appl. Microbiol., 1974; 27: 452-456.

29. Dai, Z., Wang, Y., Sun, L., Shi, Q., Xu, D.1, Wang, Y., Hu, H., Ou-Yang, Y., Lv, G. Cultivation condition and extraction method to prepare T-2 toxin using Fusarium poae. J. Microbiol., 2011; 5.

30. Cuero, R.G. Ecological distribution of F. solani and its opportunistic action related to mycotic keratitis in Cali, Colombia. J. Clin. Microbiol., 1980; 12: 455-461.

31. Ajayi, A.A., Ubon – Israel, N.N., Olasehinde, G.I. Detection of extracellular enzymes in microbial isolates from diseased carrot (Daucus carota) fruits. Int. J. Biotechnol., 2011; 2: 244-249.

32. Mishra, B.K., Dadhich, S.K. Production of amylase and xylanase enzymes from soil fungi of Rajasthan. J. Adv. Dev. Res., 2010; 1(1): 21-23.

33. Jahangeer, S., Khan, N., Jahangeer, S., Sohail, M., Shahzad, S., Ahmad, A., Khan, S.A. Screening and characterization of fungal cellulases isolated from the native environmental source. Pak. J. Bot., 2005; 37: 739-748.

34. Kwon, H.W., Yoon, J.H., Kim, S.H., Hong, S.B., Cheon, Y., Ko, S.J. Detection of extracellular enzymes activities in various Fusarium spp. Microbiology., 2007; 35: 162-165.

35. Alp, S., Arikan, S. Investigation of extracellular elastase, acid proteinase and phospholipase activities as putative virulence factors in clinical isolates of Aspergillus species. J. Basic Microbiol. 2008; 48: 331–337.

36. Friedrich, J.H., Gradisar, D.M., Chaumont, J.P.


Screening fungi for synthesis of keratinolytic enzymes. Lett. Appl. Microbiol., 1999; 28: 127-130.

37. Sharma, M., Sharma, M., Rao, V.M. In vitro biodegradation of keratin by dermatophytes and some soil keratinophiles. Afr. J. of Biochem. Res., 2011; 5: 1-6.

38. Rhodes, J.C., Bode, R.B., McCuan – Kirsch, C.M. Elastase production in clinical isolates of Aspergillus. Diagn. Microbiol. Infec. Dis., 1988; 10: 165-170.

39. Maotaza, M.S., Amany, L.K., Gadallah, M.A. Optimization of extracellular lipase production by Fusarium oxysporum. Arab. J. Biotechnol., 2005; 8: 19-28.

40. Rapp, P. Production, regulation and some property of lipase activity from F. oxysporum f. sp. Vasinfectum. Enzyme Microbiol. Technol., 1995; 9: 832-838.

41. Ch, L., Ch, C., Chen, T. Production of Acinetobacter redioresistens lipase using tween 80 as the carbon sources. Microb. Technol., 2001; 29: 258-263.

42. Abbas, H., Hiol, A., Deyris, V., Comean, L. Isolation and characterization of extracellular lipase from Mucor spp. strain isolated from palm fruit. Enzyme Microb. Technol., 2002; 31: 968-975.

43. Barletta, J.P., Angella, G., Balch, K.C., Dimova, H.G., Stern, G.A., Moser, M.T., Van Setten, G.B., Schultz, G.S. Inhibition of psedomonal ulceration in rabbit corneas by a synthetic metalloproteinase inhibitor. Invest. Ophthalmol Vis. Sci., 1996; 37: 20-28..

44. Fliesler, S.J. Lipids and lipid metabolism in the eye. J. Lipid Res., 2010; 51: 1-3

45. Shri Kant, Srivastava, D., Mishra, R.N. Toxic effects of aflatoxins on eyes – an experimental clinic-histopathological evaluation. Indian J.

Ophthalmol., 1984; 32: 242-6.46. Leema, G., Kaliamurthy, J., Geraldine, P.,

Thomas, P.A. Keratitis due to Aspergillus flavus: Clinical profile, molecular identification of fungal strains and detection of aflatoxin production. Mol. Vis., 2010; 16: 843-854.

47. Naiker, S., Odhay, B. Mycotic keratitis: profile of Fusarium species and their mycotoxins. Mycoses, 2004; 47: 50-56.

48. Raza, S.K., Mallet, A.I., Howell, S.A., Thomas, P.A. An in-vitro study of the sterol content and toxin production of Fusarium isolates from mycotic keratitis. J. Med. Microbiol., 1994; 4: 204-208.

49. Kosalec, I., Pepeljnjak, S., Jandrlic, M. Gliotoxin production by Aspergillus fumigatus strains. Arh. Hig. Rada. Toksikol., 2005; 56: 269-273.

50. Fente, C.A., Ordaz, J.J., Va´zquez, B.I., Franco, C.M., Cepeda, A. New additive for culture media for rapid identification of aflatoxin-producing Aspergillus strains. Appl. Environ. Microbiol., 2001; 67: 4858-4862.

51. Lin, M.T., Dianese, J.C. A coconut-agar medium for rapid detection of aflatoxin production by Aspergillus spp. Phytopathology., 1976; 66: 1466-1499.

52. Davis, N.D., lyer, S.K., Diener, U.L. Improved method of screening for aflatoxin with a coconut agar medium. Appl. Environ. Microbiol., 1987; 63: 1593-1595.

53. Cotty, P.J. Simple fluorescence method for rapid estimation of aflatoxin levels in a solid culture medium. Appl. Environ. Microbiol., 1988; 64: 274-276.

54. Naim, N., Saad, R.R., Naim, M.S. Production of lipids and sterols by Fusarium oxysporum (Schlecht) - Utilization of some agro-industrial by-products as additives and basal medium. Agri.Wastes., 1985; 14: 207-220.

extracellular enzymes and mycotoxins as virulence factors...

Documents