Inhibitory Effects of Antifungal Drugs on Biofilm Producing Aspergillus spp. Recovered from Drinking Water System

Research Article

J Bacteriol Mycol. 2020; 7(2): 1128.

Inhibitory Effects of Antifungal Drugs on Biofilm Producing Aspergillus spp. Recovered from Drinking Water System

Nazir NUA1*, Siddique A2, Khan MN1 and Ishaque M1

¹University of Agriculture, Faisalabad, Pakistan

²Atta -ur- Rahman School of Applied Biosciences, NUST, Islamabad, Pakistan

*Corresponding author: Noor ul Ain Nazir, University of Agriculture, Faisalabad, Pakistan

Received: April 20, 2020; Accepted: May 07, 2020; Published: May 14, 2020


Aims: Biofilms formed in drinking water distribution systems serve as a continuous source of fungal infections. Biofilms are thick aggregates of adherent microorganisms including pathogenic species of fungi. Respiratory diseases and skin allergy reactions are caused by drinking water containing biofilm forming fungus and bacteria. One of the main causes of nosocomial infections and respiratory diseases in hospitals is due to the fungal biofilm formation in machines, catheters and other surgical instruments. There are some antifungal drugs which are used to control biofilm formation to minimize the infection rate.

Methodology and results: The present study was conducted to isolate and identify Aspergillus species which are the main fungal spp. responsible for the biofilm formation in drinking water and to check their antifungal susceptibility against antifungal drugs. The isolated fungal samples from drinking water were cultivated on Potato dextrose agar for the isolation of Aspergillus species. Isolated Aspergillus species were identified on the basis of cultural, morphological and microscopic examination. Then in-vitro ability of biofilm produced by isolated Aspergillus species was estimated using microtitre plate method and quantification by crystal violet assay. Antifungal susceptibility testing against isolated fungal spp. was done by antifungal drug Amphotericin B.

Results: From results, it is concluded that drinking water of labs, hospitals and common water chillers were more prevelant by Aspergillus species whereas water from reverse osmosis plants showed negative results. From microtitre plate method and crystal violet assay, it was concluded that Aspergillus spp. are Susceptible against Amphotericin B drug as compared to miconazole.

Keywords: Aspergillus spp; Biofilm; Drinking water; Disk diffusion method; Amphotericin B


Quality of drinking water is of great importance for water consumers. One of the supreme concerns for the water consumers is contamination of drinking water with pathogenic microorganisms. These microbes include bacteria, viruses, protozoa and fungi, which are the causes of various water borne diseases and epidemics [1].

It is suggested that the occurrence of prominent fungal contaminations has been prevailing since the 1980s which causes increase in the number of immunocompromised patients such as patients of AIDS, tumor and individuals with chemotherapy treatment to get infections more easily. Furthermore, those microbes or fungus which thought to have low pathogenic potential have been found to bring about serious systemic ailment in immunocompromised hosts [2].

It is stated that fungal biofilms in a municipal water distribution system. Interpretation by scanning electron microscope proved that spores not hyphae were the primary source of possible propagates. The density level of filamentous fungi and yeasts showed that filamentous fungi were more common than yeasts in the formation of biofilm in drinking water. More established genera included Aspergillus, Alternaria, and Penicillium etc [3]. The biofilm developed inside the lining of water pipes mostly consists of opportunistic pathogens. Due to biofilm formation in water, different changes have been observed such as corrosion, water discoloration, and change in taste, water color and blockages of pipelines. The assessment of biofilm development inside of pipelines is challenging [4]. The estimation of fungi development in water, culturing techniques has been used. Moreover, high throughput sequencing techniques also used for the detection of biofilm development in water at advanced stages [5]. Fungus released its toxins in the water which causes various skin and respiratory diseases [6]. It is proposed that different factors affect the formation of biofilm. These factors includes hydraulic conditions, temperature and backwashing regime. Due to pathogenicity and enzymatic activities the chances for fungi to breakdown complex material and anthropogenic substances has increased. Fungus has bad effect on plants, invertebrates and microorganisms by producing mycotoxins. Taste and odor of water changed due to presence of fungi in it [7]. The biofilm infections are mostly relates to the skin and respiratory tract infections. In hospitals, patients who are immunocompromised are mostly at the edge of getting infections. Most of the hospitals infections occurred due to the biofilm of Aspergillus spp. whereas Aspergillus fumigatus is responsible for 90% infections [8]. Water is the basic entity on earth for life of every organism. They analyzed that drinking water in mineral bottles and tap water contaminated with different types of yeasts and filamentous fungi and found their relation with bacteria found in fecal samples. Different filter technique was used for yeasts and fungi. These were isolated on Sabouraud dextrose agar [9]. It is important to discuss the importance and applications of biofilm. Structurally biofilms are made up of microcolonies of different microbial cells (bacteria, yeasts, fungi, algae etc.) Which is 15% by volume and 85% constitutes the matrix material. The role of biofilm in microbial communities such as protection from environment, nutrient availability, acquisition of new genetic trait and penetration of antimicrobial agents was also studied [10].

In water environment, biofilm development has crucial significance. Microbes and fungus which live in biofilm are more resistant to antifungal agents and antibiotics. They are more likely to cause diseases. Biofilms mostly made bond of microbes to stones and some hard places [11].

This study was aimed to investigate the presence of Aspergillus spp. in different drinking water sources, evaluate biofilm potential of isolated Aspergillus spp. and estimation of antifungal drugs (amphotericin B and miconazole) effect against biofilm producer Aspergillus spp.

Materials and Methods

Sample collection

A total 138 drinking water samples were collected from different areas of the Faisalabad city. 80/138 samples were collected from different drinking water sources such as water chillers, Hostels, Canteen water, Laboratories of University of Agriculture Faisalabad, 10/138/ samples from the reverse osmosis plants and 48/138 samples were collected from District Headquarters and Allied Hospital, Faisalabad. Water samples were collected in sterilized plastic bottles and transferred to the laboratory of Microbiology, University of Agriculture Faisalabad. Samples were preserved at 4°C for further processing.

Isolation and Identification of Aspergillus spp

For isolation of Aspergillus, water samples were spread on Potato Dextrose Agar (PDA) plates with the help of spreader. Then plates were incubated at 37°C for 5 days in incubator. After 5 days, inoculated plates were checked for fungal growth. For identification, fungal growth appeared on the Potato Dextrose Agar plates were analysed by microscopic and macroscopic examination. For the microscopic examination, slide culture technique was used while macroscopic examination was done on the basis of morphology of fungal growth.

Spore suspension

Spores of each isolate were harvested from 7 days old pure culture in Malt Extract Agar (MEA) by swirling 2mL of saline solution (0.85%) into plates. The spore suspension was suspended and vortexed before quantification. The suspensions were standardized by dilution with saline solution. Final concentrations of 105 spores/ml were taken. Spores were counted by using a Neubauer counter chamber.

Biofilm development

Two isolated colonies were taken from the petri plates of the desired culture with sterilized loop and inoculated on Potato dextrose agar. Cultures were incubated overnight at 37°C. 100 μl of culture was taken and added in the fresh PDA broth to make the volume of 10 ml with dilution 1:10. Each diluted culture of 100 μl of both Aspergillus spp. taken added in the microtitre plates. Then, 50 μl of the distilled water and 100 μl of Potato Dextrose Agar (PDA) added in the microtitre plate respectively. Two microtitre plates contain suspension of Aspergillus fumigatus and Aspergillus flavus each were incubated for 5 days at 37°C. Biofilm formation was clearly seen after 5 days.

Microscopy examination

Biofilm formation was visualized under compound microscope and stereomicroscope. The magnification of stereo microscope and compound microscope used were 4.5X and 1000X respectively. After microscopic examination, growth medium was discarded and micro titer plates were washed twice with 200 μl of 0.85% normal saline to remove loosely attached fungus residues. Plates were air dried at 30°C for 30 minutes.

Crystal Violet Assay

Biofilm containing wells in microtitre plate were stained with 200 μl of 0.1% crystal violet for 10 minutes at room temperature. Excess dye was removed by washing each well three times with 0.85% normal saline. Then, 200 μl of 33% (v/v) acetic acid added in all wells. 100 μl of acetic acid from all wells was transferred to new plate. Optical Density (OD) of micro titer was measured by microtitre plate reader at wave length of 450 nm.

Antifungal susceptibility testing by disk diffusion method

Evaluation of antifungal susceptibility against antifungal drugs was done by Disk diffusion method. Disks of same size were taken, dip them in the Amphotericin B drug solution and placed in the plates containing Aspergillus fumigatus and Aspergillus flavus growth. Results were seen after 2 days.


Sample collection

Out of 138 samples, 87 samples were positive for Aspergillus species, of which 35 samples were Aspergillus flavus and 52 samples were Aspergillus fumigatus.

Identification of Fungi

Fungi were identified on the basis microscopic and macroscopic examinations such as colony morphology, spore structure and conidia structure. Initially, Aspergillus species were identified on the basis of morphological characteristics such as colony texture, colony appearance, and color and conidia structure. Color of the fungal colonies was blackish and brownish whereas slide culture technique showed the structure of fruiting bodies of fungus. The images were visualized under light and compound microscope at 100 and 1000 X magnification respectively. On the basis of microscopic and macroscopic examination, the fungus species were identified as Aspergillus fumigatus and Aspergillus flavus. Figure 1 and 2 shows the macroscopic examination, growth of Aspergillus spp. on Potato dextrose agar and Malt extract agar.