Investigations on anti-Aspergillus properties of bacterial products
G.L. Sharma, Institute of Genomics and Integrative Biology, Mall Road, University Campus, Delhi-110007, India
Aims: To investigate the anti-Aspergillus properties of bacterial products.
Methods and Results: In the present study, 12 bacterial strains were screened for antifungal activity against Aspergilli. The culture supernatant and lysates of Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli (BL21, DH5α, HB101, XL Blue), Klebsiella pneumoniae, Streptomyces thermonitrificans, Streptococcus pneumoniae, Enterobacter aerogenes, Staphylococcus aureus and Salmonella typhi were examined for antifungal activity in protein concentration ranging from 1000·0 to 7·8 μg ml−1 using microbroth dilution assay. The lysate of Salm. typhi and E. coli BL21 exhibited the maximum activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Their in vitro minimum inhibitory concentrations (MICs) were found to be 15·6–31·2 μg ml−1 by microbroth dilution and spore germination inhibition assays. In disc diffusion assay, a concentration of 3·1 μg disc−1 of Salm. typhi lysate showed significant activity against Aspergilli. Escherichia coli BL21 exhibited similar activity at 6·2 μg disc−1. The work on identification of molecule endowed with antimycotic properties is in progress.
Conclusion: The products of Salm. typhi and E. coli demonstrated significant activity against Aspergillus species.
Significance and Impact of the Study: This is the first time that E. coli has been reported for anti-Aspergillus activity. It could be an important source of biologically active compounds useful for developing better new antifungal drugs/or probiotics.
Infections caused due to Aspergillus species are an emerging cause of morbidity and mortality in a variety of immunocompromised patients, despite profound environmental protection and the widespread prophylactic use of agents with anti-Aspergillus activity (Richardson and Kokki 1998). The high mortality rate results also from shortcomings of the currently available therapeutic arsenal. Amphotericin B, flucytosine and itraconazole are associated with low success rates and are hampered by serious infusion- or drug-related toxicity, by hazardous drug–drug interactions, by pharmacokinetic problems and by the development of resistance (Georgopapadakou and Walsh 1996). It is, therefore, imperative to find new antifungal compounds that are not toxic to mammalian cells.
The work on bacterial species is emphasized for isolating less toxic bioactive molecules including antifungals (Woo et al. 2002). The antimicrobial activity was found to be enhanced by a pool of bacteria (Furtado et al. 2002). Various products of Pseudomonas species were reported to be active against different types of fungi, i.e. Pythium ultimum, Rhozoctonia solani, Phytophthora capsici and Fusarium oxysporium (Kim et al. 1999), Aspergillus niger (Lee et al. 1994) and Candida spp. (Sorensen et al. 1996). Further, bacillomycin and fungicin M-4 have been isolated from Bacillus species with less toxicity (Landy et al. 1948). The Nikkomycins, nontoxic to mammalian cells, were isolated from Streptomyces tendae and studied for their antifungal activities (Bormann et al. 1999). The antifungal activity spectrum of Lactobacillus coryniformis (subsp. coryniformis strain Si3) was investigated for activity against Aspergillus fumigatus (Magnusson and Schnürer 2001). The strain had strong inhibitory activity in dual-culture agar plate assays against the moulds A. fumigatus, Aspergillus nidulans, Penicillium roqueforti, Mucor hiemalis, Talaromyces flavus, Fusarium poae, Fusarium graminearum, Fusarium culmorum and Fusarium sporotrichoides. There are also bacterial products, which have been recommended for treatment of fungal diseases but their clinical efficacy is not well documented (Matricardi et al. 2003). Supplements of bacteria, probiotics, can be taken orally to refresh and complement the bacteria present in the gut. Therefore, the present study was aimed to screen the anti-Aspergillus activity of 12 bacterial strains, mostly those found in upper and lower airway/and or digestive tract.
Materials and Methods
Pseudomonas aeruginosa (MTCCB 741), Bacillus cereus (MTCCB 1272), Streptomyces thermonitrificans (MTCCB 1824), Streptococcus pneumoniae (MTCCB 655), Enterobacter aerogenes (MTCCB 111), Staphylococcus aureus (MTCCB 737), Klebsiella pneumoniae (MTCCB 109), Salmonella typhi (MTCCB 733) and Escherichia coli BL21(MTCCB 1678), DH5α (MTCCB 1652), HB101 (MTCCB 82) were procured from Institute of Microbial Technology, Chandigarh (India) and XL Blue was purchased from Banglore Genei (India). These strains were cultured in LB broth for 3 days at 37°C in a shaker incubator. The cells were counted by the turbidometry method and used for performing various experiments.
Clinical isolates of Aspergillus species, obtained from Mycology Department, Vallabhbhai Patel Chest Institute, Delhi (India) were employed in the current study. Three pathogenic species of Aspergillus namely, A. fumigatus, Aspergillus flavus and A. niger were cultured in laboratory on Sabouraud dextrose agar plates. The plates were inoculated with stock cultures of A. fumigatus, A. flavus and A. niger and incubated in a BOD incubator (Calton, New Delhi, India) at 37°C. The spores were harvested from 96 h cultures and suspended homogeneously in phosphate buffer saline (PBS), pH 7·4 in a tube. A homogeneous spore suspension was obtained by incubating the tube at 37°C for 60 min with intermittent shaking. The spores in the suspension were counted and their number was adjusted to 1 × 108 spores ml−1 before performing the experiments.
Preparation of bacterial supernatant and lysate
The 72-h log phase cultures of bacterial strains were centrifuged at 3256 g for 30 min. The supernatant was collected in a flask for testing the antifungal activity and pellet was washed with PBS thrice and suspended in sonication buffer (50 mmol l−1 Tris–HCl, 50 mmol l−1 EDTA, 5 mmol l−1 DTT, 1 mmol l−1 PMSF). The cell suspension was sonicated at 20 s bursts at 200 W and 10 s cool period using a sonicator (Misonix, Sonicator 3000; Fermingdale, NY, USA). The sonicate was centrifuged at 16 350 g for 30 min using Sorvall RC 5C centrifuge (Newtown, CT, USA). The supernatant was collected and used as lysate. The lysate was dialysed against distilled water at 4°C for 24 h with several changes of water and lyophilized. Protein concentration of bacterial supernatant and lysate was determined by BCA method of Smith et al. (1985). The lysate proteins having antifungal activity were subjected to SDS-PAGE using 12·50% gel. SDS-PAGE was carried out according to the method of Laemmli and Favre (1973).
The antifungal activity of bacterial components was analysed by microbroth dilution, disc diffusion and spore germination inhibition assays (SGIA) as described earlier (Rajesh and Sharma 2002). Each experiment was repeated at least three times.
Microbroth dilution assay. The test was performed in 96-well culture plates (Nunclon, Nunc, Myriad Industries, San Diego, CA, USA). Autoclaved Sabouraud dextrose broth (90 μl) was added to the wells of culture plates. Various concentrations of bacterial products in the range of 1000·0–3·9 μg ml−1 were prepared in the wells by twofold dilution method and these wells were inoculated with 10 μl of spore suspension containing 1 × 106 spores. The plates were incubated at 37°C and examined macroscopically after 48 h for the growth of Aspergillus mycelia. Appropriate control wells treated with amphotericin B or without any treatment were included in the study. A product was considered to be active if the wells appeared clear without any visible growth of Aspergillus and the results were expressed as minimum inhibitory concentration (MIC).
Disc diffusion assay. The disc diffusion test was performed in radiation sterilized Petri plates of 10·0 cm diameter (Tarsons, Kolkata, India). Different concentrations ranging from 25·0 to 1·56 μg of proteins/disc of the bacterial products were impregnated on the sterilized discs (5·0 mm in diameter) of Whatman filter paper No. 1. These discs were placed on the surface of the agar plates, which were already inoculated with Aspergillus spores. The plates were incubated at 37°C and examined after 48 h for zone of inhibition, if any, around the discs. The diameter of zone of inhibition was recorded. The concentration, which developed the zone of inhibition of at least 6·0 mm diameter, was considered as MIC. Amphotericin B was used in assay as a standard control drug. An additional control disc without any sample but impregnated with equivalent amount of solvent was also used in the assay.
Spore germination inhibition assay. The Aspergillus species were grown on Sabouraud dextrose agar plates and their homogenous spore suspension was prepared in the Sabouraud dextrose broth. Various concentrations (1000–3·9 μg ml−1) of the bacterial proteins in 90 μl of culture medium were prepared in 96-well flat bottom microculture plates (Nunclon, Nunc) by double dilution method. The wells were prepared in duplicates for each concentration. The wells were inoculated with 10 μl of spore suspension containing 100 ± 5 spores. Appropriate control wells treated with amphotericin B or without any treatment were included in the study. The plates were incubated at 37°C for 16 h and then examined for spore germination under inverted microscope (Nikon Diaphot, Tokyo, Japan). The number of germinated and nongerminated spores was counted and the percent spore germination inhibition (PSGI) was calculated using following formula:
The activity of the preparations was represented as the MIC90 which inhibit the germination of spores in the range of 90–100%.
The antifungal activity of supernatant and lysates prepared from P. aeruginosa, B. cereus, S. thermonitrificans, Strep. pneumoniae, Ent. aerogenes, Staph. aureus, Salm. typhi, and BL21, DH5α, HB101 and XL Blue strains of E. coli was evaluated by microbroth dilution assay. The lysate and supernatant of these strains showed mild to moderate activity. The products of Salm. typhi and E. coli demonstrated significant activity against Aspergillus species. The Salm. typhi showed highest activity against A. fumigatus (Table 1). It was observed that the protein concentration of 15·6 μg ml−1 of Salm. typhi and 31·2 μg ml−1 of BL21 lysate inhibited the growth of A. fumigatus and A. flavus completely in microbroth dilution and SGIA (Figs 1 and 2). A higher concentration of bacterial lysate of Salm. typhi and BL21 was required to inhibit the growth of A. niger. In disc diffusion assay, a concentration of 3·1 μg disc−1 of Salm. typhi lysate proteins showed significant activity against A. fumigatus and A. flavus. Escherichia coli BL21 exhibited similar activity against these species at 6·2 μg disc−1 (Tables 2 and 3). The lysate of Salm. typhi and E. coli (BL21) were run on 12·5% SDS-PAGE to find out the protein profile of these preparations. The results of SDS-PAGE showed various protein bands in these lysates in the molecular weight range of 14–98 kD. It could be useful to fractionate these proteins and identify the active antifungal fraction(s).
Table 1. Antimycotic activity of bacterial products by microbroth dilution assay against Aspergillus fumigatus
|MTCCB 1678||Escherichia coli (BL21)||62·5||31·2|
|MTCCB 1652||E. coli (DH 5α)||62·5||62·5|
|MTCCB 82||E. coli (HB 101)||62·5||125·0|
|BN Genei||E. coli (XL Blue)||125·0||250·0|
|MTCCB 1824||Streptomyces thermonitrificans||62·5||125·0|
|MTCCB 109||Klebsiella pneumoniae||125·0||125·0|
|MTCCB 741||Pseudomonas aeruginosa||31·2||125·0|
|MTCCB 655||Streptococcus pneumoniae||125·0||–|
|MTCCB 111||Enterobacter aerogenes||–||250·0|
|MTCCB 737||Staphylococcus aureus||62·5||–|
|MTCCB 733||Salmonella typhi||31·2||15·6|
|MTCCB 1272||Bacillus cereus||125·0||125·0|
Table 2. Activity of lysate of Escherichia coli (BL21) and Salmonella typhi by disc diffusion method
|25·0||11·0 ± 0·0||0·0||11·0 ± 0·0||0·0||10·8 ± 0·3||2·6|
|12·5||10·8 ± 0·3||2·6||10·7 ± 0·3||2·7||10·2 ± 0·3||2·8|
|6·2||10·3 ± 0·3||2·8||10·3 ± 0·3||2·8||7·2 ± 0·6||8·1|
|3·1||6·7 ± 0·8||11·4||7·0 ± 0·3||4·1|| 0·0||0·0|
|1·5|| 0·0||0·0|| 0·0||0·0|| 0·0||0·0|
|E. coli (BL21)|
|25·0||10·8 ± 0·3||2·6||11·0 ± 0·5||2·6||10·5 ± 0·0||0·0|
|12·5||10·5 ± 0·0||0·0||10·5 ± 0·0||0·0||6·5 ± 0·5||7·7|
|6·2|| 6·5 ± 0·5||7·7||6·5 ± 0·5||7·7|| 0·0||0·0|
|3·1|| 0·0||0·0|| 0·0||0·0|| 0·0||0·0|
|1·5|| 0·0||0·0|| 0·0||0·0|| 0·0||0·0|
|2·5||8·3 ± 0·6||7·2||8·2 ± 0·6||7·3||8·6 ± 0·5||5·8|
Table 3. MICs of lysate proteins of Escherichia coli (BL21) and Salmonella typhi by various antifungal susceptibility tests against Aspergillus species
The products of E. coli (BL21) in the present study demonstrated significant activity against Aspergilli (MIC against A. fumigatus, 31·2 μg ml−1). Matricardi et al. (2003) reported E. coli to have medicinal value. It has also been found to be useful in treating severe pseudomembranous colitis (Georg and Schlorer 1998). Such properties of E. coli may be associated with its biologically active protein molecules as indicated by observations made during current investigation. The results showed that proteins of Salm. typhi also had potential against Aspergillus species (MIC against A. fumigatus 15·6 μg ml−1). There has been no work so far on the antifungal potential of Salm. typhi. Several other products isolated from different natural sources including bacteria (Selitrennikoff 2001) have been reported to be active against fungal pathogens. Some of these antifungals were nontoxic to the hosts (Molina et al. 1993); however, a number of others had high toxicity which limited their usefulness (Lehrer et al. 1993). The antifungal activity observed in nonpathogenic bacteria such as E. coli, therefore, may be of great significance as these strains could be the source of nontoxic bioactive molecules.
Moderate type of activity was observed in S. thermonitrificans. The results revealed that the products recovered in supernatant and lysates of S. thermonitrificans had variable degree of activity. The supernatant of S. thermonitrificans showed better anti-Aspergillus activity (MIC, 62·5 μg ml−1) than lysate (MIC, 125 μg ml−1). Woo et al. (2002) studied the antifungal properties of Streptomyces supernatant containing secretory proteins and reported appreciable level of activity in supernatant; however, they did not study the Streptomyces lysates for antimycotic properties.
The products of Pseudomonas species were reported to be active against different types of fungi, i.e. Pythium ultimum, Rhozoctonia solani, Phytophthora capsici and F. oxysporium (Kim et al. 1999). In our study also, we found the supernatant of Pseudomonas to be better when compared with its lysate but high toxicity of Pseudomonadal preparations reported by Sorensen et al. (1996), may be the major limitation to work with.
The secretory proteins of B. cereus have been tested earlier as antifungals but their lysate was not used (Latoud et al. 1986). In our study we did not find significant activity in lysate or supernatant of B. cereus. The lysate and supernatant of Strep. pneumoniae, Ent. aerogenes, Staph. aureus and K. pneumoniae were found to be less active against A. fumigatus.
Although, the results of present study showed that the lysate of Salm. typhi had significant activity against Aspergilli, it was pertinent to consider E. coli BL21 lysate more important as this organism occurs as common commensal in humans. The protein profile of E. coli BL21 lysate showed various bands when run on SDS-PAGE. Such profiles will be useful for further work on identification and preparation of the active antifungal fraction(s). It may be indeed possible to develop probiotics using E. coli strains or the nontoxic formulations containing bioactive molecules of the organisms to treat various pathological conditions including mycosis.
A panel of 12 bacterial strains was investigated for activity against pathogenic fungi. The lysate of different strains of bacteria showed variable degree of antimycotic activity, MIC of Salm. typhi being the highest (15·6 μg ml−1). However, E. coli may be of greatest interest because this organism occurs as common commensal in humans. The lysate of E. coli BL21 having a mixture of proteins had potential against pathogenic isolates of A. fumigatus, A. flavus and A. niger (MIC, 31·2 to 62·5 μg ml−1). Therefore, it could be an important source of biologically active and less toxic compounds useful for developing better new antifungal preparations.
The authors wish to thank Council of Scientific and Industrial Research (CSIR), India for providing the necessary funding.