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CROSS-SPECIES INDUCTION OF ANTIMICROBIAL COMPOUNDS

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CROSS-SPECIES INDUCTION OF ANTIMICROBIAL COMPOUNDS

Cross-Species Induction of Antimicrobial Compounds

Curr Microbiol (2011) 62:974980 DOI 10.1007/s00284-010-9812-1

    Cross-Species Induction of Antimicrobial Compounds, Biosurfactants and Quorum-Sensing Inhibitors in Tropical Marine Epibiotic Bacteria by Pathogens and Biofouling Microorganisms

    Devendra H. Dusane • Pratiek Matkar • Valayam P. Venugopalan • Ameeta Ravi Kumar Smita S. Zinjarde

    Received: 20 July 2010 / Accepted: 2 November 2010 / Published online: 18 November 2010 Ó Springer Science+Business Media, LLC 2010

    Abstract Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions was investigated. Four marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and Serratia marcescens V1) displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. The marine epibiotic bacteria when co-cultivated with the aforementioned fungi or bacteria showed induction or enhancement in antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in most of the marine isolates after co-cultivation. Biosurfactant activity was signi;cantly increased when cells of B.

    pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in the presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has

    D. H. Dusane Á P. Matkar Á A. R. Kumar Á S. S. Zinjarde (&) Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411 007, India e-mail: smita@unipune.ernet.in V. P. Venugopalan Biofouling and Bio;lm Processes Section,

    Water and Steam Chemistry Division, BARC Facilities, Kalpakkam 603 102, India

    important ecological and biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite

production.

    Introduction Microbial interactions in environmental settings form the basis of several important ecological processes. Among such interactions, competition between species plays a signi;cant role in the structuring of microbial communities [16]. Microbial interactions involve the production of chemicals such as antimicrobials, surfactants and quorumsensing (QS) signal molecules, which have important commercial and medical applications [11]. Several microorganisms have developed resistance toward antibiotics and biocides. There is, therefore, a need to explore new methods and strategies for sourcing bioactive compounds. In the recent years, marine ecosystems have been exploited for the isolation of novel bioactive compounds [4]. In such ecosystems, epibiotic bacteria growing on the surfaces of marine plants and animals are known to produce compounds that exhibit antimicrobial activity. The symbiotic association of biological systems with microorganisms producing secondary metabolites prevents the settlement of potential competitors [15]. These secondary metabolites include a variety of antibiotics, biosurfactants or QS inhibitors, whose production is often governed by interactions among microbial species. Quorum sensing (QS), the phenomenon of communication between microorganisms takes place by means of small diffusible chemical signal molecules. These molecules trigger a response in target cells when a speci;c population density is reached and control several

    biological

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processes such as bioluminescence, bio;lm formation [8], production of antibiotics,

    biosurfactants [11], expression of virulence factors and interaction of bacterial populations with their eukaryotic hosts [6]. When QS molecules from one species or genus interact with those of another, they may induce or inhibit some biological processes [17]. Such an interaction is referred to as cross-species or cross-genera induction or inhibition. There is a report on cross-species inducing antibiotic production in Lactobacillus plantarum NC8 [1]. In this study, we have examined selected tropical marine epibiotic isolates on their potential to enhance production of (1) antimicrobial compounds (2) biosurfactants and (3) QS inhibitors by interaction with a set of target bacteria and fungi. The objective of this study was therefore (1) to isolate marine epibiotic bacteria producing antimicrobial compounds (2) induce or maximize the production of these bioactive compounds by exposing them to selected human pathogenic as well as marine fouling bacteria and fungi (inducer strains). We report an induction or enhancement of the aforementioned activities in the marine bacteria by pathogenic or biofouling bacteria and fungi and speculate on

the ecological and biotechnological signi;cance of such an induction.

    sequences. The sequences have been submitted to GenBank, and accession numbers have been obtained. Inducer Cultures Pseudomonas aeruginosa PA (bio;lm-forming human

    pathogen) and Bacillus pumilus BP (biofouling marine strain, GenBank Accession No: FJ938166) were the bacterial strains that were used as inducers. Two fungal strainsCandida albicans ( CA), a bio;lm forming dental isolate; and Yarrowia

    lipolytica (YL), a bio;lm forming tropical marine strain [10], were used. The bacteria and fungi were inoculated in LuriaBertani (LB) and yeast extract, peptone, dextrose

    (YPD) broth (HiMedia, India), respectively,and incubated for 24 h at 30?C. Induction

    of Marine Bacteria with Live Inducer Cultures The marine isolates were grown in 20 ml LB medium for 12 h at 30?C. Pre-grown (12 h) inducer fungi (C. albicans CA or

    Y. lipolytica YL) or bacteria (Ps. aeruginosa PA or B. pumilus BP), each containing 1 9 108 cells ml-1, were individually added (10 ll) to the asks containing the marine

    isolates (D1, S3, S8, or V1). These asks were further incubated at 30?C for 24 h.

    Flasks containing inducer cultures or epibiotic marine bacteria individually were used as controls for the experimentation. After the incubation period, cell-free supernatants (CFS) were subjected to further analysis following the methodology reported earlier [17]. Preparation of Cell-Free Supernatants (CFS) The CFS of marine bacterial isolates grown in LB, either alone or in presence of the inducer cultures were obtained by centrifugation at 10,000 rpm for 10 min at 4?C. CFS were passed through 0.22-lm ;lters (Millipore, USA), and the ;ltrate was assayed for

    antimicrobial, biosurfactant and QS inhibitory activity. Determination of Antimicrobial Activity The ability of the CFS of marine bacterial isolates to inhibit the growth of indicator microorganisms was assayed by using the standard paper disk method. The bacteria (Ps. aeruginosa PA or B. pumilus BL) and fungi (C. albicans CA or Y. lipolytica YL) were grown in LB or YPD broth, respectively, as mentioned earlier and were used as test cultures against which the antimicrobial activity was determined. The antimicrobial activity was determined on respective agar surfaces (LB or YPD). Sterile ;lter paper disks (HiMedia, India) were impregnated with 10 ll of the CFS. Paper disks were placed on agar plates previously

    Materials and Methods Isolation of Epibiotic Bacteria Epibiotic bacteria were isolated from the surfaces of the green mussel, Perna viridis and the coral, Symphyllia sp. collected from the nearshore regions of Kovalam (12?780 N, 80?250 E) and Mandapam (9?280 N, 79?120 E), Tamil Nadu, India, respectively. For the isolation of the epibiotic marine bacteria, P. viridis and Symphyllia sp. surfaces were washed with sterile seawater to remove loosely bound bacteria. The surfaces were swabbed with sterile cotton buds, re-suspended in sterile sea water, suitable dilutions were plated onto the marine agar [25] surface and the plates were incubated at 30?C for 48 h. The isolated colonies displaying distinct colony characteristics were transferred to sterile agar slants and tested for their antimicrobial activity toward Pseudomonas aeruginosa PA, Bacillus pumilus BP, Candida albicans CA and Yarrowia lipolytica YL. Identi;cation of Epibiotic Bacteria Four epibiotic isolates

    (D1, S3, S8, and V1) displaying antimicrobial activity were chosen for further studies. They were identi;ed based on analysis of their 16S rDNA

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    spread with the indicator strains and the plates were incubated at 30?C for 24 h. The zones of inhibition were measured and compared with the controls (CFS of the individual cultures). Determination of Biosurfactant Activity Using Drop Collapse Assay Drop collapse assay was performed by using the method described earlier [3] with a few modi;cations. Briey, microtiter plate lids were coated with DPX mount

    (HiMedia, India) and allowed to dry for 10 min to ensure uniform surface coating. CFS (10 ll) were placed on the coated surface and 5 ll crystal violet stains (0.2% w/v) was added to aid the visualization of the drop collapse. If the drop collapsed within 1 min, the test was said to be positive. SDS (sodium dodecyl sulfate, 1%) and growth media individually were used as control for comparison of the drop collapse. The diameter of the drop collapse was measured and compared with controls. Quorum-Sensing Inhibition (QSI) Assay Quorum-sensing inhibition (QSI) was assayed by using the indicator strain of Chromobacterium violaceum 12472 [19]. C. violaceum was grown in LB broth and was incubated for 12 h at 30?C. After the incubation period, the culture was spread on LB agar and paper disks impregnated with 10 ll of the CFS were placed on the agar surface. These plates were incubated at 30?C for 24 h. After

    incubation, the area around the paper disk showing a zone of clearance was swabbed with sterile cotton buds and streaked onto fresh agar surface to differentiate growth inhibition fromQSI. Zone of clearance with only pigment inhibition and growth of C. violaceum on fresh agar surface suggestedQSI [19]. Statistical Analysis All experiments were performed in triplicates and the data presented here are mean values. The error bars indicate standard deviation from mean values. Student’s t-test was

    used to compare sets of data and values were considered statistically signi;cant when

    P B 0.05. Data was analyzed by using Origin software version 6.0.

    viridis were identi;ed as S3: Bacillus sp., D1: Bacillus licheniformis, and S8: B. pumilus. V1: Serratia marcescens was isolated from the surface of Symphyllia sp. GenBank accession numbers obtained from NCBI are S3: HM572333; S8: HM572332; D1: HM060311; V1: GQ214001. Inherent Antimicrobial Activity Displayed by the Four Marine Epibiotic Bacteria CFS of Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and S. marcescens V1 were assayed for antimicrobial activity against Ps. aeruginosa PA, B. pumilus BP, C. albicans CA, and Y. lipolytica YL. The marine bacteria S3, D1, and V1 displayed antimicrobial activity against Ps. aeruginosa PA, B. pumilus BP, and C. albicans CA. S8 did not display any antifungal activity (Fig. 1b). None of these

    epibiotic bacteria, when cultured alone, showed any antifungal activity against Y. lipolytica YL., Bacillus sp. S3, and B. pumilus S8 displayed the highest antimicrobial activity against the pathogen, Ps. aeruginosa PA (Fig. 1a, b). B. licheniformis D1 showed antifungal activity against the pathogen, C. albicans CA as well as antibacterial activity against B. pumilus BP and Ps. aeruginosa PA (Fig. 1c). S. marcescens V1 showed greatest zone of inhibition against the biofouling B. pumilus BP. This culture also inhibited growth of Ps. aeruginosa PA and C. albicans CA (Fig. 1d). It was also observed that none of the inducer cultures (Ps. aeruginosa PA, B. pumilus BP, C. albicans CA and Y. lipolytica YL) showed antimicrobial activity against each other. Induction of Antifungal Activity by Co-cultivation The four marine epibiotic bacteria (S3, S8, D1, and V1) were co-cultivated with the pathogens or biofouling species (PA, BP, CA, or YL). Induction or enhancement of antifungal activity was analyzed using CFS. Figure 1a shows the effect of co-cultivation of Bacillus sp. S3 with the four test cultures (PA, BP, CA, or YL). From Fig. 1a, it is evident that Bacillus sp. S3 cell-free supernatant did not show activity against Y. lipolytica YL on its own. However, induction of antifungal activity was observed when cells of Bacillus sp. S3 were co-cultivated with Ps. aeruginosa PA, B. pumilus BP, or C. albicans CA. B. pumilus S8 showed induction of antifungal activity toward both Y. lipolytica YL and C. albicans CA (Fig. 1b). The antifungal activity toward Y. lipolytica YL was evident when cells of B. pumilus S8 were co-cultured with Ps. aeruginosa PA. Against C. albicans CA, the antifungal activity was also observed when cells of B. pumilus S8 were grown in association with Ps. aeruginosa PA or with C. albicans. Figure 1c shows the induction of antifungal

    Results Identi;cation of Marine Epibiotic Bacteria The marine isolates were identi;ed on the basis of 16S rDNA sequence analysis. The isolates from the surface of Perna

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    D. H. Dusane et al.: Antimicrobials, Biosurfactants and Quorum-Sensing Inhibitor Induction Fig. 1 Antimicrobial activity shown by the marine strains a Bacillus sp., S3 b Bacillus pumilus S8, c Bacillus licheniformis D1, d Serratia marcescens V1 against the pathogenic and biofouling cultures determined either individually or in association with the inducer cultures as mentioned in materials and methods section. Signi;cant enhancement in antibacterial activity is mentioned with * and antifungal activity with **

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(a)

Inhibition zone (mm)

25 20 15 10 5 0

    Ps. aeruginosa

B. pumilus

C. albicans

    Y. lipolytica

**

**

S3

S3 + PA

S3 + BP

S3 + CA

S3 + YL

(b)

Inhibition zone (mm)

    25 20 15 10 5 0

    Ps. aeruginosa

B. pumilus

C. albicans

    Y. lipolytica

* **

S8

S8 + PA

S8 + BP

S8 + CA

S8 + YL

(c)

25

    Ps. aeruginosa

B. pumilus

C. albicans

    Y. lipolytica

Inhibition zone (mm)

*

    20 15 10 5 0

D1 D1 + PA D1 + BP D1 + CA D1 + YL

*

**

(d)

25

    Ps. aeruginosa

B. pumilus

C. albicans

    Y. lipolytica

*

Inhibition zone (mm)

20 15 10 5 0

V1 V1 + PA V1 + BP V1 + CA V1 + YL

    activity in B. licheniformis D1 against Y. lipolytica YL. The cells of B. licheniformis D1 showed antifungal activity toward Y. lipolytica YL when co-cultivated with Ps. aeruginosa PA or Y. lipolytica YL. The induction of antifungal activity was maximal in case of Bacillus sp. S3 (P \ 0.05) as compared to other marine isolates when grown in association with the pathogenic and biofouling strains. From the above results the induction of antifungal

    activity was evident in the marine isolates due to co-cultivation with either the pathogens or biofouling cultures. Enhancement of Antibacterial Activity The antibacterial activity was observed with most of the marine isolates toward the pathogenic Ps. aeruginosa PA and the biofouling B. pumilus BP. Enhancement of

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antibacterial activity was less as compared with antifungal activity (Fig. 1ad).

    Antibacterial activity against Ps. aeruginosa PA was signi;cantly increased only in

    case of B. licheniformis D1 (P \ 0.05), when the cells were grown in association with Ps. aeruginosa PA (Fig. 1c). In this case, the zone of inhibition increased from 13 mm to 17 mm, after induction with Ps. aeruginosa PA. When S. marcescens was co-cultured with Ps. aeruginosa PA, an increase in antibacterial activity toward B. pumilus was observed. The zone of inhibition increased from 20 to 22 mm (Fig. 1d). The results suggest cross-genera and cross-species induction and enhancement of antimicrobial activity in epibiotic marine isolates when confronted by pathogenic or biofouling cultures. Enhancement of Biosurfactant Production by Co-cultivation An enhancement in biosurfactant production was assessed when the marine isolates were co-cultivated with the pathogens or biofouling cultures. As described earlier, CFS were analyzed by the drop collapse assay and the zone diameters of collapse drop were measured. When cells of B. pumilus S8 were grown in association with the pathogen Ps. aeruginosa PA or biofouling strain of B. pumilus BP, there was enhanced drop collapse. The zone diameter of the drop collapse increased in a statistically signi;cant manner (P \

    0.05) when compared to S8 or BP individually. Similar enhancement in biosurfactant activity was also observed when B. licheniformis D1, was grown in association with B. pumilus BP or C. albicans CA (Table 1) The zone diameter of the drop collapse increased when D1 was co-cultivated with B. pumilus BP or with C. albicans CA. S.

marcescens V1 showed enhancement in

    biosurfactant activity when co-cultivated with the biofouling strain of B. pumilus BP. The zone diameter increased from 5.5 mm (S. marcescens V1 alone) to 7.5 mm (S. marcescens V1 co-cultivated with B. pumilus BP). The difference in drop collapse was statistically signi;cant (P = 0.001). The results suggest signi;cant enhancement in

    biosurfactant activity when S. marcescens V1 was co-cultivated with the biofouling B. pumilus culture as compared with other marine isolates. Quorum-Sensing Inhibition (QSI) In order to determinethe QSI activity of marine isolates we compared their ability to reduce pigment production in the indicator strain of C. violaceum 12472. The marine Bacillus sp. S3 when grown in association with Ps. aeruginosa PA showed inhibition in pigment production of C. violaceum 12472 (Fig. 2a). QSI in this case was, however, exhibited to a lower extent than that observed with Ps. aeruginosa PA alone (Fig. 2b). The other marine isolates did not show production of quorum-sensing inhibitor compounds either alone or when co-cultivated with inducer cultures.

    Discussion Recent discoveries have suggested the role of quorum sensing as an important mechanism used by bacteria to compete with one another and to produce secondary metabolites. We followed a method described earlier [17] to

    Table 1 Enhanced biosurfactant production by culture supernatants of marine bacterial isolates, pathogenic, and biofouling cultures either individually or in association with inducer cultures as estimated by the drop collapse assay Surfactant source SDS PA S3 ? YL S8 ? PA S8 ? BP D1 ? BP D1 ? CA V1 ? BP Zone diameter (mm) 6.0 8.0 6.1 7.0 7.0 6.5 7.1 7.5 Fig. 2 Quorum-sensing inhibition of indicator Chromobacterium violaceum 12472 culture displayed by the cell-free supernatant of (a) Bacillus sp. S3 when grown in association with Ps. aeruginosa PA (b) with Ps. aeruginosa PA individually

    SDS sodium dodecyl sulfate, PA Pseudomonas aeruginosa, S3 Bacillus sp. S3, YL Yarrowia lipolytica, S8 Bacillus pumilus S8, BP Bacillus pumilus BP, D1 Bacillus licheniformis, CA Candida albicans, V1 Serratia marcescens

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    check the ability of living pathogenic and biofouling fungi and bacteria to induce or enhance antimicrobial, biosurfactant, andQSI activities. Chemical interactions between different species of bacteria can affect the production and secretion of antimicrobial secondary metabolites [5]. For example, quorum-sensing molecules

    (homoserine lactones) produced by several bacterial species are known to induce the biosynthesis of the antibiotic phenazine in Ps. aeruginosa [24] and stimulate the production of carbapenem by Erwinia carotovora [23]. There are thus a few reports on induction of antibacterial activity. However, combined study on induction of antimicrobial activity, biosurfactant production, andQSI by pathogenic bacteria and fungi has not been investigated earlier. In these studies by the authors, the antimicrobial activity of epibiotic bacteria against pathogenic and biofouling fungi and bacteria was induced or enhanced when they were exposed to the inducer strains. The antifungal activity was maximally induced after exposure of pathogenic and biofouling fungi and bacteria to the non-producer marine bacteria. Antibacterial activity was observed against the pathogenic Ps. aeruginosa PA and the biofouling B. pumilus BP. The results suggest cross-species and crossgenera induction and enhancement of antimicrobial activity in marine isolates when co-cultivated with the indicator cultures. This thus emphasizes the importance of community level interactions between microorganisms from two different ecological niches. It was suggested that the enhanced production of bioactive compounds may occur due to a competition for nutrients and space and to inhibit the potential competitors [20]. The extent of induction of bioactive property could vary due to response of signal molecules between communities and survive against potential competitors [5]. These pathogenic bacteria may not be the natural competitors for the epibiotic bacteria and therefore lack the secondary metabolites that would induce the antimicrobial activity [17]. Biosurfactants are also important bioactive compounds produced by microbial cells. They are known to reduce the surface and interfacial tension. The marine epibiotic bacterial isolates studied here belonged to the genus Bacillus and Serratia. These results complement the earlier reports on both these species producing such surfactants. Bacillus sp. is known to produce lipopeptide group of biosurfactants such as lichenysin and surfactin [9]. Serratia marcescens is a known producer of serrawettin and sucrose-lipid biosurfactants [22]. In this study, the inducer culture Ps. aeruginosa PA displayed maximal biosurfactant activity. The role of quorum-sensing molecules in rhamnolipid production has been recently reviewed [11]. Biosurfactant synthesis was enhanced when cells of the inducer B. pumilus BP were added to the marine epibionts S. marcescens V1, B. pumilus S8 and B. lichenifomis D1. C. albicans CA also

    increased biosurfactant production in B. licheniformis D1, possibly due to quorum sensing. QSI is one of the important mechanisms to control virulence in some microbial systems [13]. It has been reported to regulate virulence in human and plant pathogens such as E. carotovora and Ps. aeruginosa [7, 21]. This may also signify a natural antimicrobial strategy utilized by plants and other organisms to control bio;lm

    formation [2]. QSI compounds thus may ;nd potential application in biofouling

    prevention. C. violaceum 12472 (the indicator of QSI used in this study) produces pigment under the control of quorum sensing [19]. This indicator bacterium regulates pigment production by the quorum-sensing molecule, N-hexanoylHSL (C6-HSL). We observed the inhibition of pigment production in C. violaceum 12472 by cell-free

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