Biotechnol Lett (2012) 34:655661 DOI 10.

1007/s10529-011-0825-z

ORIGINAL RESEARCH PAPER

Extracellular protease in Actinomycetes culture supernatants inhibits and detaches Staphylococcus aureus biolm formation
Joo-Hyeon Park Jin-Hyung Lee Chang-Jin Kim Jae-Chan Lee Moo Hwan Cho Jintae Lee

Received: 25 October 2011 / Accepted: 2 December 2011 / Published online: 11 December 2011 Springer Science+Business Media B.V. 2011

Abstract Bacterial biolms are associated with chronic infections due to their resistance to antimicrobial agents. Staphylococcus aureus is a versatile human pathogen and can form biolms on human tissues and diverse medical devices. To identify novel biolm inhibitors of S. aureus, the supernatants from a library of 458 Actinomycetes strains were screened. The culture supernatants (1% v/v) of more than 10

Actinomycetes strains inhibited S. aureus biolm formation by more than 80% without affecting the growth. The culture supernatants of these biolmreducing Actinomycetes strains contained a protease (equivalent to 0.1 lg proteinase K ml-1), which both inhibited S. aureus biolm formation and detached pre-existing S. aureus biolms. This study suggests that protease treatment could be a feasible tool to reduce and eradicate S. aureus biolms. Keywords Actinomycetes Biolm Protease Staphylococcus aureus

Electronic supplementary material The online version of this article (doi:10.1007/s10529-011-0825-z) contains supplementary material, which is available to authorized users.

Introduction
J.-H. Park J.-H. Lee M. H. Cho J. Lee (&) School of Chemical Engineering, Yeungnam University, Gyeongsan, Republic of Korea e-mail: jtlee@ynu.ac.kr J.-H. Park e-mail: 20721381@ynu.ac.kr J.-H. Lee e-mail: jinhlee@ynu.ac.kr M. H. Cho e-mail: mhcho@ynu.ac.kr C.-J. Kim e-mail: changjin@kribb.re.kr J.-C. Lee Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea e-mail: jclee777@kribb.re.kr

In natural niches, bacteria grow in polymicrobial communities. Hence, competition or cooperation between the community members is important for bacterial survival in limited resources and space. As a survival strategy, many bacteria form biolms that are sessile microbial communities attached to a surface by polysaccharides, proteins and nucleic acids. Biolms exhibit reduced sensitivity to conventional antibiotics, host defenses, and external stresses and contribute to bacterial persistence in chronic infections (Costerton et al. 1999; Hoffman et al. 2005). Staphylococcus aureus is an important human pathogen that often exhibits antibiotic resistance. It is responsible for worldwide outbreaks of nosocomial infections (Lowy 1998). Biolm formation is one of

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the mechanisms of antibiotic resistance in S. aureus (Stewart and Costerton 2001). Therefore, it is important to understand how S. aureus controls its biolm so as to discover non-toxic compounds capable of inhibiting or dispersing biolms without allowing bacteria to develop drug resistance. The diverse mechanisms and environmental conditions that contribute to biolm formation and disassembly of S. aureus include quorum sensing, protease, DNase, cis-2-decenoic acid, D-amino acids such as D-leucine, D-methionine, D-tyrosine, and D-tryptophan, phenol-soluble polypeptides, and pH change (Boles and Horswill 2011). Particularly, the protease in S. aureus inhibited its own biolm formation and dispersed established biolms via agr quorum-sensing system (Vuong et al. 2000; Boles and Horswill 2008). A serine protease in S. epidermidis inhibited S. aureus biolm formation and nasal colonization (Iwase et al. 2010). Some Actinomycetes contain anti-biolm compounds that are active against the biolms produced by several pathogenic bacteria. As examples, an extract from Streptomyces albus inhibited biolm formation of Vibrio harveyi (You et al. 2007), and methanol extracts of coral-associated S. akiyoshiensis inhibited biolm formation of S. aureus (Bakkiyaraj and Pandian 2010). However, no further information on these anti-biolm compounds and their mechanisms of biolm reduction has been reported. The goal of this study was to identify potent biolm inhibitors from the high-throughput screening of an Actinomycetes library against S. aureus. The Actinomycetes library has been established for the discovery of antibiotics, anti-inammatory drugs, and anti-cancer compounds. A library of 458 Actinomycetes strains was screened to identify novel biolm inhibitors against S. aureus and found that the culture supernatants of more than 10 Actinomycetes strains signicantly inhibited S. aureus biolm formation and triggered its biolm dispersal through a high activity of protease.

that were obtained from Korean Agricultural Culture Collection. The Actinomycetes library, constructed from soil Actinomycetes by the Microbial Resource Center in Korean Institute of Bioscience and Biotechnology (Daejeon, Korea), was used to screen the biolm inhibitors. Initially, Actinomycetes strains isolated from soil in South Korea were maintained at 28C for 7 days on Bennetts agar plates containing 10 g glucose, 1 g yeast extract, 2 g peptone, 1 g beef extract and 15 g agar per liter (pH 7.0). All Actinomycetes strains were cultured for the production of active compounds under the same conditions in glucose/soluble starch (GSS) medium (10 g soluble starch, 20 g glucose, 25 g soybean meal, 1 g beef extract, 4 g yeast extract, 2 g NaCl, 0.25 g K2HPO4, and 2 g CaCO3 per liter at pH 7.2). Supernatants of Actinomycetes cultures were stored at 4C until used. Bacterial culture A single colony of S. aureus was inoculated in LB (25 ml) in 250 ml asks and cultured at 37C and 250 rpm. Overnight cultures were re-inoculated at 1:100 in fresh medium. Growth was monitored from the OD600 values which were converted into dry cell weights (OD600 of 1 = 240 mg/l). Each experiment was performed with at least two independent cultures.

16S rRNA gene sequencing Genomic DNA was prepared as previously described (Tamaoka and Komagata 1984). The 16S rRNA gene was amplied by PCR with the forward primer Eubac 27F and the reverse primer 1492R (DeLong 1992). Direct sequence determination of the PCR-amplied DNA was carried out using an automated DNA sequencer (ABI 3730XL, Applied Biosystems). The 16S rRNA gene sequences were compared with available sequences from GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) to determine approximate phylogenetic afliations. Sequence similarity values were computed using the EzTaxon server (http://www.eztaxon.org/; Chun et al. 2007). Crystal violet biolm assay

Materials and methods Bacterial strains and chemicals All experiments were conducted at 37C, and Luria Bertani (LB) medium was used for the culture of S. aureus (ATCC 25923) and S. aureus (ATCC 6538)

A static biolm formation assay was performed in 96-well polystyrene plates (see Pratt and Kolter 1998).

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657
250

Briey, cells initially at OD600 of 0.05 were grown for 24 h without shaking at 37C. Total biolm formation was measured at 570 nm using crystal violet. For dispersion assay, S. aureus was initially cultured in 96-well plates for 7 or 14 h without shaking at 37C. Supernatants of Actinomycetes strains were added and the cultures were incubated for 7 or 17 h more before assaying the biolm. The supernatants of Actinomycetes strains were ltered through a 0.45 lm lter to remove bacteria before adding in the culture of S. aureus. Each data point was averaged from at least three independent cultures. Determination of protease activity Proteolytic activity was determined using skim milk agar plates (Quiblier et al. 2011) containing 5 g of non-fat dry milk and 0.5 g Bacto-agar (Difco) in 50 ml distilled water. To detect the extracellular protease activity, supernatants (10 ll) of Actinomycetes strains were added in a hole in the milk/agar plates and incubated at 37C after 24 h. GSS medium (10 ll) and proteinase K (Sigma-Aldrich Co.) were used as negative and positive controls, respectively. Protein digestion was observed by a clear zone surrounding Actinomycetes supernatants.

Number of Actinomycetes strains

208
200

150

100

92 77 65 16

50

0 5-20 20-40 40-60 60-150 150-200

Biofilm formation

Fig. 1 Histogram of biolm modulation in S. aureus by the Actinomycetes library. The biolm screening of S. aureus (ATCC 25923) was performed with the supernatants (1%, v/v) of each Actinomycetes strain in 96-well plates at 37C after 24 h. The numbers on the top of each bar the number of Actinomycetes strains. The biolm formation (%) in the X-axis represents the relative biolm formation (100 9 biolm formation with the supernatant of Actinomycetes species/biolm formation of untreated control)

culture supernatants of soil Actinomycetes strains primarily reduced S. aureus biolm formation. Identication of active Actinomycetes strains 16S rRNA gene sequencing was utilized to identify 10 Actinomycetes strains having the highest antibiolm activity. BLAST searching against the GenBank database revealed a list of diverse Actinomycetes strains (Table 1). Nine of the strains were Streptomyces genus and one was Kribbella genus. For further study, the two most active supernatants of Streptomyces strains (AN090250 and AN091562) were chosen and we named as Streptomyces sp. BFI 250 and the strain Kribbella sp. BFI 1562. More detail characteristics of two strains are described in Supplementary material. Low concentration of Actinomycetes supernatants inhibited S. aureus biolm formation without reducing the growth of planktonic S. aureus cells Supernatants of Streptomyces sp. BFI 250 and the strain Kribbella sp. BFI 1562 clearly and dosedependently inhibited the biolm formation of two S. aureus strains (ATCC 25923 and ATCC 6538)

Results Screening the Actinomycetes library for inhibiting S. aureus biolm formation To identify the antibiolm compounds against S. aureus (ATCC 25923), the supernatants from 458 soil Actinomycetes strains were screened. The supernatants were used at 1% (v/v) without any growth reduction of S. aureus cells greater than 40% of the nal cell density compared with an untreated control, although one supernatant of Actinomycetes strain showed antimicrobial activity (no growth) on S. aureus cells. The antibiolm screening demonstrated various abilities to control the biolm formation of S. aureus (Fig. 1). Seventy seven Actinomycetes strains reduced the biolm formation of S. aureus by more than 80%, while 16 Actinomycetes strains increased the biolm formation by more than 50%, as veried by repeating the screening more than three times. Therefore, the

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658 Table 1 List of 10 Actinomycetes strains with a high antibiolm activity against S. aureus ID Strains Sequence identity (%) 100 100 99 99 99 99 99 99 99 99

Biotechnol Lett (2012) 34:655661

Biolm formation reduction (%) 82 8 95 4 95 1 92 6 92 8 92 8 87 5 83 4 93 7 94 4

Relative protease activity ?? ??? ??? ? ? ? ? ?? ? ???

AN090185 AN090230 AN090250 AN090282 AN090374 AN090410 AN090414 AN090423 AN090426 AN091562

Streptomyces sp. HB096 Streptomyces hebeiensis NBRC 101006 Streptomyces anulatus NRRL B-2000T Streptomyces laceyi Streptomyces turgidiscabies strain HBUM174876 Streptomyces griseoruber Streptomyces akiyoshiensis strain HBUM174525 Streptomyces sp. DA10202 Streptomyces lanatus Kribbella hippodromi

Active Actinomycetes strains were identied by 16S rRNA gene sequencing and the names of the closest strains are shown with sequence identity (%). IDs represent arbitrary numbers in Actinomycetes library. Biolm formation reduction is the relative reduction of the biolm formation of S. aureus (ATCC 25923) in the presence of Actinomycetes culture supernatants compared to the biolm formation of S. aureus with GSS medium as a control. Relative protease activity represents a protease activity compared to the activity of proteinase K

(Fig. 2a, b, e). Even a very low concentration (0.01%) of Streptomyces sp. BFI 250 and Kribbella sp. BFI 1562 inhibited the biolm formation of S. aureus strain (ATCC 25923) by more than 80% (Fig. 2a, b). However, the supernatants of Streptomyces sp. BFI 250 and Kribbella sp. BFI 1562 were not toxic to S. aureus cells (Fig. 2c). These results indicated that the biolm reduction of S. aureus by Actinomycetes supernatants could be due to their antibiolm activity, but not due to their antimicrobial activity. High protease activity in the supernatants of Actinomycetes strains was responsible for the biolm reduction of S. aureus To identify the component in the supernatants of Actinomycetes that was responsible for the biolm reduction in S. aureus, solvent extraction was initially performed using various solvents such as methanol, hexane, ethyl acetate, ethyl ether, and chloroform. However, none of the solvent extracts showed any antibiolm activity while the water residue after solvent extraction contained the antibiolm activity (data not shown). Furthermore, heat treatment of the supernatants at 100C for 10 min completely eliminated the antibiolm activity of the supernatants (Fig. 2a, b). These results suggested that the active antibiolm components in the supernatants were some peptides or proteins.

As protease activity plays an important role in S. aureus biolms (Boles and Horswill 2011), the addition of proteinase K (0.1 lg ml-1) clearly reduced the biolm formation of S. aureus by more than 80% (Fig. 2d). Therefore, protease activity assay was performed using milk agar plates. Apparently, the supernatants of Streptomyces sp. BFI 250 and Kribbella sp. BFI 1562 showed a high protease activity (clear circle zone by degrading milk) as a positive control proteinase K showed a high protease activity (Supplementary Fig. 1). The amount of protease in the supernatants of Streptomyces sp. BFI 250 and Kribbella sp. BFI 1562 corresponded to approx. 0.1 lg proteinase K ml-1. In addition, all 10 Actinomycetes strains showed the protease activity to different extents (Table 1), while 16 biolm-inducing Actinomycetes strains (see Fig. 1) did not show any protease activity (data not shown). Hence, the culture supernatants of Actinomycetes strains produce large amounts of extracellular protease which inhibits biolm formation of S. aureus. Therefore, the extracellular protease appears to be a major antibiolm component in the supernatants of Actinomycetes strains. Supernatants of Actinomycetes strains triggered biolm detachment of S. aureus Like biolm development, dispersion of established biolms is important in biolm control. Hence, the

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a
Biofilm formation (OD570)
0.6 0.5 0.4 0.3 0.2 0.1 0.0
0

Biofilm formation (OD570)

ATCC 25923

b
0.6 0.5 0.4 0.3 0.2 0.1 0.0
0

ATCC 25923

c
2.0

ATCC 25923

Dry cell weight (g l -1)

1.5 1.0 0.5 0.0

0 5 10 15 20 25 30

None BFI 250 BFI 1562

0.001 0.01 0.1

1.0

Heat

0.001 0.01 0.1

1.0

Heat

BFI 250 supernatant (%, v/v)

BFI 1562 supernatant (%, v/v)

Time (h)

d
Biofilm formation (OD570)
0.6 0.5 0.4 0.3 0.2 0.1 0.0
0

Biofilm formation (OD570)

ATCC 25923

e
2.0 1.5 1.0 0.5 0.0

ATCC 6538
None 0.1 1

0.001 0.01 0.1

1.0

Heat
-1

Proteinase K ( g ml )

Prot K -1 (g ml )

BFI 250 BFI 1562 (%, v/v) (%, v/v)

Fig. 2 The supernatants of Streptomyces sp. BFI 250 and Kribbella sp. BFI 1562 reduced S. aureus biolm formation. Two strains of S. aureus (ATCC 25923 and ATCC 6538) were used and the total biolm formation (OD570) of S. aureus was measured in the presence of the supernatants at 37C after 24 h

in 96-well plates (a, b, and e). The cell growth of S. aureus (ATCC 25923) was measured in the presence of the supernatants of Actinomycetes strains (1%, v/v) at 37C and 250 rpm (c). Proteinase K as a control was added in the beginning of the culture for biolm assay (d)

biolm dispersal ability was investigated with the supernatants of Actinomycetes strains. As with the treatment using proteinase K, the culture supernatants of Actinomycetes strains signicantly inhibited and dose-dependently disassembled pre-existing biolms of S. aureus (Fig. 3). Specically, the supernatants of Streptomyces sp. BFI 250 and Kribbella sp. BFI 1562, at 0.01% (v/v) detached the established S. aureus biolms by more than 80% over 17 h. Similarly, a shorter dispersion time (7 h rather than 17 h shown in Fig. 3) after adding the supernatants of Streptomyces sp. BFI 250 and a longer culture time (14 h instead of 7 h) before adding the Actinomycetes supernatants showed almost the same result of biolm dispersion (Supplementary Fig. 2). Therefore, the supernatants of Actinomycetes inhibited and dispersed S. aureus biolms.

Discussion Chronic S. aureus infections are frequently associated with biolm formation. The genetic mechanism of

S. aureus biolm formation is complex and the subject of much research. Among the diverse mechanisms of biolm formation and disassembly (OGara 2007; Boles and Horswill 2011), extracellular proteases play an important role in biolm formation and disassembly in S. aureus and also proteinase K from other species inhibited biolm formation and dispersed preexisting biolm via the agr quorum-sensing system in S. aureus (Boles and Horswill 2008). Furthermore, a serine protease from S. epidermidis was recently inhibited S. aureus biolm formation and nasal colonization (Iwase et al. 2010). In accord with previous studies, the present study demonstrates that a high protease activity from Actinomycetes signicantly reduced the biolm formation and enhanced the biolm dispersal of S. aureus. Unlike most antibiotics that primarily aim to inhibit cell growth, this biolm inhibitor did not affect cell growth and offers a reduced chance of developing resistance (Hentzer et al. 2002; Lesic et al. 2007). In nature, bacteria tend to grow in polymicrobial communities and prefer to live in the biolm state (Sauer et al. 2004). It is likely that Actinomycetes

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a
Biofilm formation (OD 570)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

ATCC 25923
None 0.001 0.01 0.1 1

In conclusion, diverse Actinomycetes strains secrete signicant amounts of an extracellular protease to inhibit biolm formation in S. aureus. Treatment with such proteases offers the possibility of controlling the antibiotic resistant biolms of S. aureus. The Actinomycetes library is an important resource of biolm inhibitors and other bioactive compounds.
Acknowledgments This research was supported by the Yeungnam University research grant. J.-H. Park was supported by the Human Resources Development Program of Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant (No:20104010100580) funded by the Korean Ministry of Knowledge Economy.

Prot K -1 ( g ml )

BFI 250 (%, v/v)

BFI 1562 (%, v/v)

b
Biofilm formation (OD 570)

3.0 2.5 2.0 1.5 1.0 0.5 0.0

ATCC 6538
None 0.001 0.01 0.1 1

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Prot K -1 (g ml )

BFI 250 (%, v/v)

BFI 1562 (%, v/v)

Fig. 3 The supernatants of Actinomycetes strains dispersed established S. aureus biolm. S. aureus ATCC 25923 (a) and ATCC 6538 (b) were cultured to form biolm for 7 h without shaking, supernatants of Actinomycetes strains were added to S. aureus cultures in 96-well plates, and the cultures were incubated for 17 h more without shaking. Total biolm formation of S. aureus was measured at OD570. Prot K proteinase K in the unit of lg ml-1 and BFI 250 and BFI 1562 supernatants of Actinomycetes strains AN090250 and AN091562, respectively, in the unit of % (v/v)

encounters S. aureus naturally since S. aureus can be found in the soil where Actinomycetes is dominant (McNeil and Brown 1994). Hence, there would be competition and defense in multispecies bacterial consortia. Actinomycetes species produce a wide variety of secondary metabolites including antibiotics, parasiticides, and herbicides, which are likely to aid their survival in the presence of other microbes, insects, and even animal hosts. It has been suggested here that Actinomycetes strains employ extracellular protease to reduce the biolm formation of another bacteria, S. aureus. In contrast, S. aureus intentionally decreases its biolm formation in order to move to other locations and nutritional sources.

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