In vitro inhibition of Aspergillus fumigatus by Candida species, especially C. albicans and C. glabrata

In vitro inhibition of Aspergillus fumigatus by Candida species, especially C. albicans and C. glabrata H. S. Randhawa*, R. S. Sandhu and T. Kowshik Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, P.O. Box No. 2101, Delhi 110 007, India In vitro inhibition of Aspergillus fumigatus by Candida albicans, C. dubliniensis, C. glabrata, C. krusei and C. tropicalis is reported here. A. fumigatus was variably inhibited by all of the five Candida species tested. At 48 h incubation, A. fumigatus was strongly inhibited by C. albicans as well as by C. dubliniensis, both in the sealed and unsealed peptone glucose agar (PGA) plates. Of all the Candida species tested, C. glabrata proved to be the strongest antagonist, with three of its isolates causing complete inhibition, whereas the fourth allowed negligible growth of A. fumigatus. Five-day-old sealed cultures of C. tropicalis and C. krusei caused about 83 and 79% growth inhibition of A. fumigatus, respectively. Irrespective of the Candida species tested, the inhibitory effect was considerably greater in sealed cultures than in the unsealed cultures, indicating that the predominant inhibitory factor was a volatile metabolite(s). The experimental data further revealed that the role of any nonvolatile metabolite(s) was a subsidiary one as the A. fumigatus inhibition attributed to them seldom exceeded 10%. It was also observed that the inhibitory effect of various Candida species on A. fumigatus was fungistatic and not fungicidal. Attention is drawn to clinical implications of the inhibition of A. fumigatus by C. albicans that may interfere with isolation of the former from sputum or other clinical specimens harbouring this yeast-like fungus as a commensal or colonizer, thereby delaying a definitive diagnosis of aspergillosis and initiation of specific antifungal chemotherapy. Pending additional investigations, the identity of the inhibitory Candida metabolites and their mode of action remained undetermined. CANDIDA albicans (Robin) Berkhout (1923), a yeast-like fungus, is widely recognized as a normal inhabitant and opportunistic pathogen of humans and animals. It can be isolated from varied sites on or in the human body, primarily the gastrointestinal tract, the oropharynx and other mucocutaneous regions 16. It is well known that C. albicans has an in vitro inhibitory effect on the growth of dimorphic systemic pathogens, Histoplasma capsulatum and Blastomyces dermatitidis, and the dermatophytes, Trichophyton mentagrophytes, T. rubrum and Epidermophyton floccosum, thereby causing difficulties in their *For correspondence. (e-mail: vpci@delnet.ren.nic.in) 860 primary isolation from clinical specimens that may be harbouring it 711. Chaturvedi et al. 12 have earlier reported in vitro antagonistic interactions between B. dermatitidis and six other human pathogenic fungi, including C. albicans. The present study was prompted by the consideration that in vitro biointeractions between various Candida species and many of the human pathogenic fungi have so far remained unexplored. In this communication, we report the in vitro inhibition of Aspergillus fumigatus Fresenius (1863), the principal etiologic agent of aspergillosis, by C. albicans, C. glabrata (H. W. Anderson) S. A. Meyer & Yarrow (1978), C. dubliniensis Sullivan, Westerneng, Haynes, Bennett & Coleman (1995), C. tropicalis (Castellani) Berkhout (1923) and C. krusei (Castellani) Berkhout (1923). A series of experiments with two-member cultures, involving one of the test Candida species and A. fumigatus were conducted. The test fungi included authentic cultures of C. albicans, J 1012, ATCC 36801, ATCC 44374, ATCC 44505, ATCC 44506, C. dubliniensis, CD 36 (Irish oral isolate from HIV + patient, and type strain of the species), C. glabrata, ATCC 90030, Y 33.90, and A. fumigatus, SP 31/2K, available in the Culture Collection, Department of Medical Mycology, V.P. Chest Institute, Delhi. C. tropicalis, MCCLS 420002, C. krusei, MCCLS 440003 and C. glabrata, B 95 and B 1011, were received through the courtesy of Dr Arunaloke Chakrabarti, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh. They were grown in peptone glucose agar (PGA, peptone, 1%; glucose, 2%; agar, 2%; ph 7.0) or broth medium incubated at 28 1 C. Inoculum of the Candida species was prepared from 48 h growth on PGA plates at 28 C. The growth was suspended in 5 ml of sterile distilled water, and its density was adjusted to McFarland standard No. 1, containing 4.5 10 6 cells/ml approximately. The conidia from a 48 to 72-h-old PGA culture of A. fumigatus, SP 31/2K, growing at 28 C were harvested in 5 ml of sterile distilled water containing 0.05% tween 80, and the suspension was adjusted to contain 1520 conidia per high power microscopic field. The inhibitory effect of various Candida species on A. fumigatus was tested in two-member pure cultures incubated in the dark at 28 C. This was done by streaking the test Candida species in a circle or semicircle/u-shaped pattern, whereas A. fumigatus was point-inoculated in the centre of the plates. For a further study of colonial interactions, the paired fungi were point-inoculated 15 mm apart with the Candida species preceding A. fumigatus by 48 h, so that they were not masked early by the latter due to its rapid growth. The controls consisted of single and paired colonies of the test A. fumigatus isolate. Three replicates were put up for each test and the experiments were repeated at least once. The colonial interactions were classified after Porter 13 as modified by Skidmore and Dickinson 14. CURRENT SCIENCE, VOL. 82, NO. 7, 10 APRIL 2002

To investigate the inhibitory role of nonvolatile and volatile metabolites of Candida species on the in vitro growth of A. fumigatus, two sets of experiments were done. In the first, each test Candida species was inoculated at the centre of a dialysis membrane strip, 50 mm 50 mm, placed on a PGA agar plate. After 5 days of incubation at 28 C, the membrane strip along with the growth was removed, followed by inoculation of A. fumi- Table 1. In vitro inhibitory effect of volatile metabolites of Candida species, cultured as circular peripheral streaks, on A. fumigatus point-inoculated in the centre of PGA plates incubated at 28 C Colony diameter of A. fumigatus after incubation period of 48 h 72 h 96 h 120 h Candida species Control: 22 mm 22 mm 37 mm 38 mm 49 mm 47 mm 59 mm 58 mm C. albicans J 1012 ATCC 36801 ATCC 44374 ATCC 44505 ATCC 44056 8 mm (78.3) 7 mm (81.0) 12 mm (67.5) 6 mm (83.7) 6 mm (83.7) 6 mm (84.2) 8 mm (78.9) 17 mm (65.3) 18 mm (63.2) 22 mm (55.1) 15 mm (69.3) 10 mm (79.5) 9 mm (80.8) 10 mm (78.7) 7 mm (85.1) 26 mm (55.9) 27 mm (54.2) 30 mm (49.1) 24 mm (59.3) 20 mm (66.1) 6 mm (89.6) 12 mm (79.3) 12 mm (79.3) 8 mm (86.2) C. dubliniensis CD-36 9 mm (75.6) 15 mm (69.3) 27 mm (54.2) C. glabrata Y 33.90 B95 B1011 ATCC 90030 C. tropicalis MCCLS 420002 6 mm (72.7) 6 mm (72.7) 18 mm (51.3) 8 mm (78.9) 25 mm (51.0) 9 mm (80.8) 34 mm (42.3) 10 mm (82.7) C. krusei MCCLS 440003 15 mm (31.8) 27 mm (27.0) 7 mm (81.5) 34 mm (30.6) 10 mm (78.7) 43 mm (27.1) 12 mm (79.3), negligible growth without any colony formation;, no growth. Figures in parentheses denote per cent inhibition of A. fumigatus growth vis-à-vis its control. Table 2. In vitro effect of nonvolatile diffusible metabolites of Candida species on growth of A. fumigatus point-inoculated at the centre of PGA plates at 28 C Colony diameter of A. fumigatus after incubation periods of 48 h 72 h 96 h 120 h Candida species Control: 22 mm 37 mm 51 mm 68 mm C. albicans J 1012 ATCC 36801 ATCC 44374 ATCC 44505 ATCC 44506 28 mm (4.5)* 22 mm ( )** 21 mm (4.5) 21 mm (4.5) 39 mm (5.4) 38 mm (2.7) 35 mm (5.4) 34 mm (8.1) 36 mm (2.7) 53 mm (3.9) 51 mm () 45 mm (11.7) 43 mm (15.6) 49 mm (3.9) 67 mm (1.4) 66 mm (2.9) 59 mm (13.2) 57 mm (16.1) 64 mm (5.8) C. dubliniensis CD-36 32 mm (13.5) 47 mm (7.8) 60 mm (11.7) C. glabrata Y 33.90 ATCC 90030 B 95 B 1011 21 mm (4.5) 36 mm (2.7) 34 mm (8.1) 35 mm (5.4) 36 mm (2.7) 49 mm (3.9) 45 mm (11.7) 48 mm (5.8) 49 mm (3.9) 65 mm (4.4) 60 mm (11.7) 60 mm (11.7) 62 mm (8.8) C. tropicalis MCCLS 420002 21 mm (4.5) 36 mm (2.7) 51 mm () 66 mm (2.9) C. krusei MCCLS 440003 35 mm (5.4) 48 mm (5.8) 64 mm (5.8) *Figures in parenthesis denote per cent inhibition of A. fumigatus growth vis-à-vis its control. **No inhibition. CURRENT SCIENCE, VOL. 82, NO. 7, 10 APRIL 2002 861

gatus at the centre of the plate. The colonial dimensions of A. fumigatus were compared with those of its control after incubation periods of 48, 72, 96 and 120 h. In the second set of experiments, two-member streak cultures of A. fumigatus were prepared together with each of the test Candida species on PGA plates. The Candida species were inoculated as a peripheral circular streak, employing a cotton-tipped sterile swab dipped in a culture suspension estimated to contain 4.5 10 6 cells/ml approximately (McFarland standard No. 1), whereas A. fumigatus was point-inoculated at the centre of the plate, using a 2-mm wide spatula needle dipped in conidial suspension prepared as already described. One replicate set of culture plates was sealed with parafilm M (American National Can TM, Greenwich, CT 06836), incubated at 28 C and observed up to 5 days. A. fumigatus inhibition was assessed by measuring the colony diameter of A. fumigatus and comparing it with that of the control. The results are presented in Tables 1 and 2 and depicted in Figures 17. The in vitro growth of A. fumigatus was variably inhibited by all the C. albicans and C. glabrata test strains, and this observation was not strain specific. At 48 h incubation, A. fumigatus was strongly inhibited by C. albicans, J 1012, ATCC 36801, ATCC 44374, ATCC 44505, ATCC 44506 and by C. dubliniensis, CD 36, both in the sealed and unsealed PGA plates. With C. albicans, ATCC 44505 the negligible growth of A. fumigatus remained unchanged even after 120 h of incubation in sealed cultures in sharp contrast to its extensive growth, attaining a colony diameter of 24 mm and 5859 mm in unsealed cultures and in the controls, respectively. C. krusei, MCCLS 440003 caused the lowest inhibition of 27% in unsealed cultures examined after 72 h and 120 h of incubation. However, it allowed only negligible growth in sealed cultures after 48 h incubation as seen in paired cultures with the various C. albicans and C. dubliniensis isolates. Inhibition due to C. tropicalis, MCCLS 420002, ranged from 42% in unsealed cultures to 83% in sealed cultures at 120 h incubation. It was noted that the inhibitory effect of C. glabrata was more marked than that of any other Candida sp. investigated. Thus, A. fumigatus was completely inhibited by C. glabrata, B 95, B 1011 and ATCC 90030, whereas isolate Y33.90 allowed negligible growth after 72 h of incubation only in unsealed cultures. Also, the inhibition of A. fumigatus was much more in the sealed than in the unsealed cultures irrespective of the antagonist test Candida spp., indicating that the predomi- a b c d Figure 1. Comparative inhibitory effect of C. albicans, C. glabrata and C. tropicalis on A. fumigatus, SP 31/2K, cultured on PGA plates incubated at 28( 1) C for 72 h. Candida species were inoculated in a semicircular/u-shaped streak, whereas A. fumigatus, SP 31/2K, was pointinoculated in the centre. a, Control; pure culture of A. fumigatus isolate showing its typical pigmented colony and unrestricted growth; b, Restricted growth of A. fumigatus isolate due to inhibition by C. albicans, ATCC 36801; c, Markedly restricted growth of A. fumigatus isolate due to inhibition by C. glabrata, Y 33.90; and d, Typical growth of A. fumigatus isolate apparently unaffected by C. tropicalis, MCCLS 420002. a b c Figure 2. Inhibitory effect of C. albicans on A. fumigatus, SP 31/2K, in circular and semicircular streak cultures on PGA plates incubated at 37 C for 72 h. a, Control; A. fumigatus colony in pure culture showing more rapid growth than at 28 C (Figure 1 a); b, Colony of A. fumigatus showing restricted growth and loss of pigmentation due to C. albicans, J 1012, inoculated as a circular streak; and c, Same as in (b), except that the restricted and unpigmented A. fumigatus colony is growing freely towards the top end away from the semicircular streak culture of the antagonist, C. albicans, J 1012. 862 CURRENT SCIENCE, VOL. 82, NO. 7, 10 APRIL 2002

a b c RESEARCH COMMUNICATIONS Figure 3. Inhibitory effect of C. albicans, J 1012 and C. glabrata, ATCC 90030 inoculated as a circular streak at the periphery of the plate on A. fumigatus, SP 31/2K cultured on unsealed PGA plates at 28 C for 72 h. a, Control; A. fumigatus colony on unsealed PGA plate; b, A. fumigatus colony showing considerable inhibition of radial growth due C. albicans, J 1012 seen as a dense circular band of smooth yeast-like growth at the periphery; and c, A. fumigatus showing negligible growth at the centre due to strong inhibitory effect of C. glabrata, ATCC 90030. a b c Figure 4. Inhibitory effect of C. albicans, J 1012, and C. glabrata, ATCC 90030, inoculated as a circular peripheral streak on A. fumigatus, SP 31/2K, cultured on sealed PGA plates at 28 C for 72 h. a, Control. A. fumigatus colony on sealed PGA plate; b, A. fumigatus colony showing marked inhibition of radial growth due to C. albicans, J 1012; and c, A. fumigatus colony showing negligible growth due to strong inhibition by C. glabrata, ATCC 90030. Note that the inhibition is more than in the sealed PGA plate (Figure 3 c). Figure 5. In vitro antagonism between C. albicans, ATCC 36801 (inoculated as a circular peripheral streak) and A. fumigatus inoculated in the centre of a sealed PGA plate incubated at 28 C for 4 weeks. Note the dark central zone (a) of A. fumigatus colony with heavy sporulation, surrounded by a dense granular sporulation zone (b), a wide clear inhibitory area with sparse sporulation (c) bound by a dense sporulation ring marking the interface with C. albicans, and a wide granular peripheral zone of dense sporulation (d) occurring all over the C. albicans growth. Increasing density of sporulation towards the centre (c to a) may be attributed to the waning effect of the inhibitory non-volatile metabolites diffusible into the culture medium. nant inhibitory factor(s) was a volatile metabolite(s). This inference was supported by additional data (Figure 7), showing that the inhibition of A. fumigatus due to nonvolatile metabolite(s) seldom exceeded 10%. Inhibition exceeding this level was seen only in pairings with C. albicans, ATCC 44374, ATCC 44505, C. dubliniensis, CD-36 and C. glabrata, ATCC 90030 and B 95 at 120 h incubation. The highest inhibition due to nonvolatile metabolite(s) recorded was 16% with C. albicans, ATCC 44505 at 96 h and 120 h of incubation. It can be inferred from these results that non-volatile metabolite(s) have only a subsidiary role in the inhibition of A. fumigatus (Figure 2 c). It is pertinent to add that the inhibition of A. fumigatus due to the various Candida species was found to be fungistatic and not fungicidal. This was tested by subculturing the inhibited growth on PGA slants incubated at 28 C for one week. Killer toxin-producing strains have been reported in several yeast genera such as Saccharomyces, Hansenula, Kluyveromyces, Pichia and Candida, including C. glabrata, C. krusei and C. guilliermondii, but their lethal effects have been studied only in yeasts 1517. There is little information on the suscep- CURRENT SCIENCE, VOL. 82, NO. 7, 10 APRIL 2002 863

tibility of aspergilli and other filamentous fungi to the killer yeast toxins. Further observations on in vitro colonial interactions between A. fumigatus and various Candida species were made by point-inoculation of the antagonists 15 mm apart on PGA plates with the Candida spp. preceding A. fumigatus by 48 h. After 96 h of incubation at 28 C, there was slight inhibition of growth and sporulation of A. fumigatus at its colonial interface with C. albicans, Figure 6. Photomicrograph of a lactophenal cotton blue mount from a 5-day-old growth of A. fumigatus cultured in the centre of a sealed PGA plate at 28 C along with C. glabrata, B 1011, inoculated as a peripheral circular streak. Note that the inhibitory effect is manifested by the formation of distorted branched hyphae with irregular, terminal or intercalary aberrant swollen structures, 230. J 1012, ATCC 36801, ATCC 44505 and C. dubliniensis, CD-36. In the paired culture with C. glabrata, Y 33.90, the interacting colonies were about 1 mm apart, with A. fumigatus showing slight inhibition of growth but not of sporulation. By 144 h of incubation, the rapidly spreading A. fumigatus had partly overrun the colonies of C. albicans, C. dubliniensis and C. glabrata. Notably, there was an arc of dense sporulation of A. fumigatus at colonial interface of the paired fungi. Upon further incubation (9 days), A. fumigatus overgrew and completely masked the colonies of the aforementioned Candida spp. In the classification of colonial bio-interactions based upon the criteria of Porter 13 as modified by Skidmore and Dickinson 14, the interactions between A. fumigatus and Candida spp. corresponded to Type III in which one of the paired fungi ceases to grow and gets overgrown by the opposing fungus. The nature of Candida inhibitory metabolite(s) and their mode of action on A. fumigatus is as yet unknown, but work is now underway to study this aspect. Previously, carbon dioxide has been reported as the inhibitory metabolite responsible for in vitro inhibition of Trichophyton mentagrophytes and some other dermatophyte species by C. albicans 18. Finally, it seems pertinent to point out that our observations on the in vitro inhibition of A. fumigatus by Candida spp., especially C. albicans, may be of clinical interest in that this commensal fungus may interfere with the isolation of A. fumigatus from sputum or other clinical specimens harbouring it as a commensal or colonizer, Figure 7. Effect of non-volatile, diffusible metabolite of Candida species on A. fumigatus cultured on PGA plates at 28 C up to 120 h. 864 CURRENT SCIENCE, VOL. 82, NO. 7, 10 APRIL 2002

thereby delaying a definitive diagnosis of aspergillosis and initiation of specific anti-fungal therapy in the patients. 1. Winner, H. I. and Hurley, R., Candida albicans J. & A., Churchill Ltd, London, 1964. 2. Odds, F. C., Candida and Candidosis, Bailliere Tindall, London, 1988. 3. Rippon, J. W., Medical Mycology. The Pathogenic Fungi and the Pathogenic Actinomycetes, W.B. Saunders Co, Philadelphia, USA, 1988, pp. 175180. 4. Kwon-Chung, K. J. and Bennett, J. E., Medical Mycology, Lea and Febiger, Baltimore, USA, 1992, pp. 280285. 5. Beneke, E. S. and Rogers, A. L., Medical Mycology and Human Mycoses, Star Publ. Co, Belmont, USA, 1996, pp. 149 160. 6. Segal, E. and Elad, D., in Topley & Wilson s Microbiology and Microbial Infections (eds Collier, L., Balows, A. and Sussman, M.), vol. 4; Medical Mycology (eds Ajello, L. and Hay, R. J.), Arnold, London, 1998, pp. 423428. 7. Nickerson, W. J. and Jillson, O. F., Mycopathol. Mycol. Appl., 1948, 4, 279283. 8. Kapica, L., Shaw, C. 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R.S.S. thanks the University Grants Commission, New Delhi, for the award of an Emeritus Fellowship and H.S.R. thanks the Indian National Science Academy, New Delhi, for the award of an Honorary Senior Scientist position. Thanks are also due to Dr V. K. Vijayan, Director, V.P. Chest Institute, and Dr H. C. Gugnani, Professor of Medical Mycology, for laboratory facilities and Vinay Kumar and S. Mazumdar for assistance. Received 8 September 2001; revised accepted 13 December 2001 Microfouling of manganese-oxidizing bacteria in Tuticorin harbour waters S. Palanichamy, S. Maruthamuthu*, S. T. Manickam and A. Rajendran # Offshore Platform and Marine Electrochemistry Centre, Central Electrochemical Research Institute Unit, Harbour Area, Tuticorin 628 004, India *Central Electrochemical Research Institute, Karaikudi 630 006, India Implication of manganese-oxidizers in corrosion of various alloys stimulated the investigators to concentrate on these aspects. In the present study, an attempt was made to bring out the bacterial genera involved in the oxidation of manganese in biofilms. The materials immersed in sea water for biofilm formation included polyvinyl chloride (PVC), stainless steel (SS), brass and copper. The biofilm samples were analysed quantitatively and qualitatively for both heterotrophic bacterial population (HB) and manganese-oxidizing heterotrophic bacterial population (MHB). Both qualitative and quantitative examination of biofilms showed relatively poor population density on copper. Qualitative examination revealed the representation of both Gram-positive and Gram-negative bacteria on all materials. However, only Gram-positive groups, especially of the endospore-forming genus Bacillus and non-endospore forming genus Propionibacterium were observed on copper coupons. Gram-positive genera dominated over Gram-negative genera in most of the biofilms studied. The genera identified under manganese-oxidizing bacterial isolates were Bacillus, Staphylococcus, Synecoccus, Propionibacterium, Micrococcus, Pseudomonas and Vibrio. Among them, Bacillus species was most commonly encountered in all the materials studied. Potential measurements for SS316 showed positive shift. Analysis revealed enormous amount of manganese in the biofilms. THE affinity of marine bacteria for surfaces was first studied by Zobell 1 who demonstrated that some bacterial cells approach a surface, adhere rapidly to it, initiate glycocalyx (exopolysaccharide) production and form the discrete microcolonies that are the basic organizational units of biofilms. Costerton and Lewandowski 2, thus defined the biofilms as a matrix enclosed bacterial populations adhere to each other and/or to surfaces or interfaces. The type and the rate of bacterial adhesion influence the nature of the surface concerned, which subsequently leads to corrosion of the material. Sulphatereducing bacteria and iron-oxidizing bacteria have long been considered as major contributors to corrosion. Recently, manganese-oxidizers have also been identified as major contributors to corrosion 35. Following the first report of Mollica and Trevis 6 to show ennoblement of # For correspondence. (e-mail: ttn-sohum@sancharnet.in) CURRENT SCIENCE, VOL. 82, NO. 7, 10 APRIL 2002 865