Keywords
Antifungal susceptibility -
Aspergillus species - biofilm - phospholipase - virulence factors
Introduction
Aspergillus species is a filamentous fungus that causes infections in immunocompetent and immunocompromised
individuals. Among the recognized species, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger are the most encountered etiological agents causing 95% of the disease. The diseases
caused by Aspergillus in humans are collectively known as aspergillosis. The infections caused by Aspergillus species can be localized, allergic, non-invasive, or invasive/disseminated infections.
The mortality rate of invasive/disseminated aspergillosis in high-risk groups varies
between 40% and 90%, based on factors such as host immune status, site of infection,
and appropriate treatment.[1]
Pathogenic Aspergillus strains require virulence factors to infect the host. Production of virulence factors
by Aspergillus species contributes to the establishment of infection, which at a later stage may
lead to invasive/disseminated infections.[2] The virulence factors of the pathogen include biofilm production, lipase, phospholipase,
amylase, hemolysin, proteinase, esterase, and elastase.[3] Biofilm formation mainly contributes to fungal virulence by promoting the adherence
of hyphae to host cells and enhancing resistance to killing by antifungals.[4] A variety of extracellular enzymes produced by Aspergillus species break down the complex polysaccharides into simple sugars, which are assimilated
and used for growth, reproduction, and the survival of the host organism.[5]
Delay in diagnosis and insufficient information on the virulence of the organism are
important contributors for the high mortality in invasive fungal infections.[6] For effective therapy and direct future research, it is also essential to comprehend
the association between virulence and antifungal susceptibility profiles of the fungus
causing invasive infections. Hence, this study was undertaken to understand the association
between production of various virulence factors and the antifungal susceptibility
profiles of the Aspergillus isolates, which will help to provide an effective treatment.
Materials and Methods
This hospital-based descriptive study was undertaken in the Department of Microbiology
in a tertiary care center after obtaining the institutional ethics clearance. For
this study, 52 Aspergillus isolates grown from samples of patients collected over 13 months (May 2018 to June
2019) were considered. Repetitive isolates from the same patients were excluded from
the study. Basic demographic details were collected from all patients. The strains
were maintained in glycerol stock media at 4°C with regular sub-culture for once in
3 months for further testing.[7]
For characterization, the Aspergillus isolates were inoculated into Sabouraud dextrose agar (SDA) plates containing gentamycin,
and incubated at 25°C for 5 days with regular examination once in 2 days. Once growth
appeared in SDA plates, their macroscopic colony morphology was observed. The isolates
were systematically identified based on their microscopic morphology on LPCB tease
mount and/or slide culture technique.
Test for Virulence Factors
Test for Virulence Factors
Aspergillus niger ATCC 6275 was the control strain used for testing all virulence factors.
Biofilm Formation
Aspergillus isolates were evaluated for biofilm production by test tube method using 0.1% crystal
violet stain.[8] The scoring for tube method was done visually and compared with the results of the
control strains. Visible film formation lining the wall and bottom of the tubes was
considered to be positive. The appearance of a ring at the liquid interface was not
considered as indicative of biofilm formation. The amount of biofilm formed was scored
as 0-negative, 1-weak, 2-moderate, and 3-high/strong. The procedure was performed
in triplicates.[9]
Preparation of Aspergillus Suspension
Fresh Aspergillus isolate (24 to 48 hours) was mixed in sterile saline and the turbidity was adjusted
to 0.5 McFarland turbidity standard. Ten microliters of suspension of each isolate
was carefully placed on the recommended test medium and incubated at 25°C for 4 to
5 days to evaluate the lipase, phospholipase, amylase, and hemolytic activities. For
enzyme production, all strains were tested three times for the enzymatic evaluation
and interpreted by two independent observers.
Lipase Activity
To determine the lipase activity, Tween 80 agar plates with the phenol red indicator
medium was used.[10] Lipase activity was considered as positive if there was a change in color from pink
to lemon yellow. Transformation of media color from reddish pink to lime yellow was
detected due to a change in the pH from basic to acidic (from pH 7.2 to below 6.5)
state, caused by the release of the fatty acid due to the degradation of Tween 80.
Phospholipase Activity
Egg yolk agar plates were used to determine the phospholipase production.[11] The phospholipase activity was marked by the presence of a dense white zone of precipitation
around the inoculum. Egg yolk metabolized by phospholipase create precipitation around
fungal colonies. The ratio between the colony diameter and the colony diameter plus
the halo zone was used to assess the enzymatic activity (Pz) as described by Price
et al. The Pz values were classified into four categories based on the scores: elevated
Pz values of 1.0 were considered negative, Pz values between 0.999 and 0.700 were
considered low enzyme activity (+), Pz values between 0.699 and 0.400 were considered
moderate enzyme activity (+ +), and Pz values between 0.399 and 0.100 for strong enzyme
activity (+ + +).
Amylase Activity
The amylolytic activity was determined using the starch agar plate method.[12] Lugol's iodine solution was added to the culture plate after incubation. A clear
zone around the inoculation site was considered as positive for amylolytic activity.
Iodine reacts with starch to develop a dark brown color. Hydrolysis of starch will
therefore form a clear zone around the inoculum.
Hemolytic Activity
Hemolytic activity was determined using a 7% sheep blood agar plate.[13] A transparent/semi-transparent zone around the inoculation site was considered as
positive for hemolytic activity. By forming pores or holes in red blood cell membranes,
hemolysin lyses red blood cells (RBCs), resulting in the release of iron that promotes
microbial growth.
Antifungal Susceptibility Testing
Antifungal Susceptibility Testing
Paecilomyces variotii Centraalbureau voor Schimmelcultures (CBS) 132734 was the control strain used for
antifungal susceptibility testing.
The antifungal susceptibility testing was done using broth microdilution method, adhering
to the CLSI M38-A2 guidelines.[14] The antifungal agents and the range of concentration tested were amphotericin B
(A9528-50MG, Sigma-Aldrich, USA): 0.125 to 16 µg/mL.
Itraconazole (16657-100MG, Sigma-Aldrich, USA): 0.125 to 16 µg/mL.
Voriconazole (PZ0005-5MG, Sigma-Aldrich, USA): 0.125 to 16 µg/mL.
Posaconazole (32103-25MG, Sigma-Aldrich, USA): 0.125 to 16 µg/mL.
The results were analyzed and interpreted according to the CLSI guidelines after 48 hours
of incubation. As a quality check, the complete absence of turbidity in the media
control well was checked each time the test was performed.
Results
Out of 52 Aspergillus isolates obtained from clinical samples, 43 (82.69%) were from ear swab, 4 (7.69%)
were from pus, 3 (5.77%) were from tracheal aspirate, and 2 (3.85%) were from bronchial
wash.
Age and Gender Distribution
Age and Gender Distribution
In this study, percentage of females (69.23%) was more than the males (30.77%), with
male to female ratio of 1:2. The age of the patients ranged between 4 and 65 years
(mean age: 43 years). Most patients were in the age group 41 to 60 years (42.31%).
Speciation of Aspergillus Isolates
Speciation of Aspergillus Isolates
The Aspergillus isolates were speciated based on their macroscopic appearance on SDA and microscopic
appearance on the LPCB mount.[15] Out of the total 52 Aspergillus isolates, 28 i.e., 53.84% isolates were identified as A. niger, which is the most predominant isolate in our study. A. flavus was the second-most prominent species isolated in 19/52 isolates, i.e., 36.54%, followed
by A. fumigatus in 5/52 isolates, i.e., 9.62%.
Production of Virulence Factors
Production of Virulence Factors
The control strain Aspergillus niger ATCC 6275 produced consistent results for all virulence factors tested.
Among the 52 Aspergillus isolates tested for biofilm production using tube method, 8 (15.38%) were strongly
positive, 12 (23.08%) were weakly positive, and 32 (61.54%) were negative. None of
the isolates tested showed a moderate positive result for biofilm production. Out
of the 52 Aspergillus isolates tested, 25 (48.08%) were lipase positive ([Fig. 1A]), and the remaining 27 (51.92%) were negative ([Fig. 1B]) for lipase activity. Phospholipase activity was present in 25 (48.08%), out of
the 52 isolates tested with a mean Pz = 0.95 ([Fig. 1C]). Among these 25 isolates which showed phospholipase activity, 15 (60%) were moderate
positive, 10 (40%) were low positive, and 27 (51.92%) were found to be negative for
phospholipase activity ([Fig. 1D]). None of the isolates tested showed a high production of phospholipase activity.
Of the 52 Aspergillus isolates, 29 (55.77%) exhibited amylase activity ([Fig. 2A]) and 23 (44.23%) were negative for amylase activity ([Fig. 2B]). Among the 52 Aspergillus isolates, 33 (63.46%) possessed hemolytic activity ([Fig. 2C]), and the remaining 19 (36.54%) were found to be negative for hemolytic activity
([Fig. 2D]).
Fig. 1 Lipase activity on Tween 80 agar medium. (A) Lipase positive (arrow showing color change from red to yellow). (B) Lipase negative. Phospholipase activity on egg yolk agar medium. (C) Phospholipase positive (arrow showing a white zone of precipitation). (D) Phospholipase negative.
Fig. 2 Amylase activity on starch agar medium. (A) Amylase positive (arrow showing a clear halo). (B) Amylase negative. Hemolytic activity on blood agar medium. (C) Hemolysis present. (D) Hemolysis absent.
Distribution of Virulence Factors among Aspergillus species
Distribution of Virulence Factors among Aspergillus species
Among the 52 Aspergillus isolates tested for virulence factors, A. niger showed the maximum production of biofilm (46.43%), lipase (89.29%), phospholipase
(57.14%), and hemolytic (82.14%) activity, whereas amylase activity was observed more
in A. flavus (89.47%). A. fumigatus did not produce any other virulence factor, except amylase activity. The distribution
of virulence factors among Aspergillus species is shown in ([Table 1]).
Table 1
Distribution of virulence factors among Aspergillus species
|
Virulence factors
|
A. niger (n = 28)
|
A. flavus (n = 19)
|
A. fumigatus (n = 5)
|
|
Biofilm
|
Positive (n = 20)
|
13 (46.43%)
|
7 (36.84%)
|
0
|
|
Negative (n = 32)
|
15 (53.57%)
|
12 (63.16%)
|
5 (100%)
|
|
Lipase
|
Positive (n = 25)
|
25 (89.29%)
|
0
|
0
|
|
Negative (n = 27)
|
3 (10.71%)
|
19 (100%)
|
5 (100%)
|
|
Phospholipase
|
Positive (n = 25)
|
16 (57.14%)
|
9 (47.37%)
|
0
|
|
Negative (n = 27)
|
12 (42.86%)
|
10 (52.63%)
|
5 (100%)
|
|
Amylase
|
Positive (n = 30)
|
9 (32.14%)
|
17 (89.47%)
|
3 (60%)
|
|
Negative (n = 22)
|
19 (67.86%)
|
2 (10.53%)
|
2 (40%)
|
|
Hemolytic
|
Positive (n = 33)
|
23 (82.14%)
|
10 (52.63%)
|
0
|
|
Negative (n = 19)
|
5 (17.86%)
|
9 (47.37%)
|
5 (100%)
|
Antifungal Susceptibility Testing
Antifungal Susceptibility Testing
For all 52 Aspergillus isolates, the mean MIC of itraconazole was found to be higher (6.53 μg/mL) than that
of voriconazole (0.93 μg/mL) and posaconazole (0.21 μg/mL) among the triazoles tested
in our study. Amphotericin B had the second-highest mean MIC value of 3.77 μg/mL.
However, the mean MIC values of A. fumigatus was less for amphotericin B (1.15 µg/mL) and itraconazole (0.15 µg/mL) when compared
to other Aspergillus species ([Table 2]). The resistance percentage was more for itraconazole (48.08%) followed by amphotericin
B (28.85%). Posaconazole had 100% sensitivity for all Aspergillus isolates. Among the 28 Aspergillus niger isolates, 3 (10.71%) isolates were found to be resistant to both itraconazole and
voriconazole, and 2 (7.14%) were found to be resistant to amphotericin B and itraconazole.
Among 19 A. flavus isolates, two (10.53%) were found to be resistant to itraconazole and voriconazole.
Table 2
Mean MIC values and range of the antifungal agents
|
Aspergillus species (n = 52)
|
Mean MIC values/range of the antifungal agents (µg/mL)
|
|
Amphotericin B
|
Itraconazole
|
Voriconazole
|
Posaconazole
|
|
A. niger
(n = 28)
|
4.69
|
3.77
(16–0.125)
|
6.96
|
6.53
(16–0.125)
|
0.99
|
0.93
(8–0.125)
|
0.26
|
0.21
(2–0.125)
|
|
A. flavus
(n = 19)
|
3.11
|
7.58
|
0.97
|
0.17
|
|
A. fumigatus (n = 5)
|
1.15
|
0.15
|
0.43
|
0.13
|
Comparison of Virulence Factors and Resistance Strains of Aspergillus Species
Comparison of Virulence Factors and Resistance Strains of Aspergillus Species
All amphotericin B-resistant isolates produced biofilm, itraconazole-resistant isolates
exhibited phospholipase activity, while voriconazole-resistant strains produced biofilm
and showed phospholipase and hemolytic activities ([Fig. 3]). Although A. fumigatus is the most common organism that causes invasive aspergillosis, it was found to be
less virulent and less resistant. However, A. niger, which is the third-most common organism causing invasive aspergillosis, was found
to produce most of the virulence factors and was highly resistant to the antifungal
agents tested ([Tables 1] and [2]). Posaconazole was found to be sensitive regardless of the virulence factors produced
by the Aspergillus species, hence comparison was not included for the same.
Fig. 3 Comparison of virulence factors and resistance strains of Aspergillus species.
Discussion
Virulence factors production among Aspergillus species could play an important role in the pathogenesis of invasive aspergillosis.[16] Treatment of such invasive infections are difficult if the organism is highly virulent
and drug-resistant. In these conditions, comparison of virulence factors and antifungal
susceptibility pattern helps the physician to start the patient with a suitable antifungal
agent.
In our study, A. niger (54%) was found to be the most predominant isolate, followed by A. flavus (37%) and A. fumigatus (9%). This finding was similar to a few other studies done by Khaled et al[17] and Satish et al,[18] where A. niger was the predominant isolate followed by A. flavus. Our finding differed from another study, where the author found A. flavus as the predominant isolate.[19] Hence, it is implied that the distribution of Aspergillus species varies by geographical location.
The biofilm production in the present study was seen only among 20/52 (38.46%) isolates.
In a previous study by Raksha et al,[20] biofilm production was more, i.e., 32/39 (82%) Aspergillus isolates produced biofilm. The test tube method with crystal violet staining could
be an acceptable biofilm quantification procedure, but has a high degree of subjective
uncertainty, as reading is taken through visual inspection and hence, does not detect
moderate to poor biofilm producers accurately.[10] Hence, a better method such as tissue culture plate which is considered to be the
gold standard for detection of biofilm production, could be adopted.
In our study, the percentage of production of the enzymes lipase 25/28(89.29%) and
phospholipase 16/28(57.14%) was more among A. niger compared to A. flavus and A. fumigatus, which is similar to the findings by Zohri et al,[21] where the maximum number of A. niger strains produced the enzymes lipase 27/30 (90%) and phospholipase 30/30 (100%). In
our study, among the Aspergillus species, A. niger had the maximum enzymatic activity which contributes to its role in establishment
of infection in susceptible individuals that may later progress to invasive infections
in such patients. In the present study, the production of amylase was maximum in A. flavus 17 (89.47%), which was in agreement with an earlier study by Fadahunsi et al,[22] whereas this was contrary to the findings of Singh et al,[23] where A. fumigatus produced the maximum amylase activity. Diverse sources of carbon and nitrogen can
affect the production of amylase.[22] In our study, hemolytic activity was more in A. niger (82.14%) followed by A. flavus (52.63%), similar to an earlier study by Mezher et al,[13] where A. niger produced the maximum hemolytic activity. Hence, in this study, Aspergillus isolates were observed to be able to produce an array of extracellular enzymes.
In our study, Aspergillus isolates showed high MICs to itraconazole followed by amphotericin B. This was in
agreement with a few other studies by Dannaoui et al[24] and Karuthaas et al,[25] where high MICs for itraconazole were observed when compared to amphotericin B.
This finding suggest that this drug may not be the right choice to treat invasive
aspergillosis. Though voriconazole had a low mean MIC value of 0.93 µg/mL, five (9.62%)
of our isolates were resistant to voriconazole. A previous study by Lestrade et al[26] also showed that among 196 patients with invasive aspergillosis 37 (19%) harbored
voriconazole-resistant infections. In another study by Sabino et al[27] and Baddley et al[28] high voriconazole mean MIC values were reported for Aspergillus isolates. Voriconazole with low mean MIC values to Aspergillus isolates can be used to treat patients with aspergillosis. But the problem with this
approach is that because there is an emergence of voriconazole resistance being documented
globally, in vitro antifungal susceptibility testing is suggested before starting
the treatment. Posaconazole with the lowest mean MIC value of 0.21 µg/mL was found
to be the most effective antifungal agent when compared to amphotericin B and other
azoles. This finding was comparable to a previous report by Sabino et al.[27] Therefore, posaconazole may be considered for the treatment of aspergillosis caused
by voriconazole-resistant Aspergillus isolates.
A major virulence attribute of Aspergillus species, other than antifungal drug resistance, is the production of biofilm that
could potentially result in treatment failure and infection recurrence. In our study,
all 15 (28.85%) amphotericin B and 5 (9.62%) voriconazole-resistant isolates produced
biofilm. These results suggest that higher doses or antifungal combination therapy
should be considered for a better penetration of drugs to fungal cells if the isolate
is a biofilm producer. Also, in our study, all itraconazole- and voriconazole-resistant
isolates exhibited phospholipase activity. In a previous study by Mohammadi et al
on Candida albicans isolates showed a significant association between high MICs of fluconazole/itraconazole
and phospholipase production.[29] Another study by Ghorbel et al had noted a high resistance percentage of amphotericin
B among high phospholipase producers of A. flavus, whereas our study did not have such correlation between amphotericin resistance and
phospholipase production.[6] The role of virulence in the disease outcome has hardly been considered in aspergillosis,
despite the data obtained for other microorganisms.[30]
Conclusion
Even though virulence factor testing for Aspergillus species has been done in many studies, no comparison of virulence factors and antifungal
susceptibility testing has been done in India. Though in our study we could establish
an association between virulence factor production and antifungal resistance, more
research with large sample size may confirm the findings. This warrants their wider
surveillance to clearly specify the exact role of virulence factors and drug resistance,
especially in patients with invasive aspergillosis. In addition, more genetic and
molecular studies may assist in enhanced understanding of the relationship between
virulence factors and antifungal resistance, which may help in designing novel therapeutic
approaches.
Note
This study was presented at MICROCON 2018 (01/12/2018).
This study was approved by the institutional ethics committee with ethics clearance
number: NI/20/FEB/74/09.
