1 23

Applied Microbiology andBiotechnology ISSN 0175-7598 Appl Microbiol BiotechnolDOI 10.1007/s00253-014-6317-z

Surfactin from Bacillus subtilis displays anunexpected anti-Legionella activity

Clémence Loiseau, MargotSchlusselhuber, Renaud Bigot, JoanneBertaux, Jean-Marc Berjeaud & JulienVerdon

1 23

Your article is protected by copyright and

all rights are held exclusively by Springer-

Verlag Berlin Heidelberg. This e-offprint is

for personal use only and shall not be self-

archived in electronic repositories. If you wish

to self-archive your article, please use the

accepted manuscript version for posting on

your own website. You may further deposit

the accepted manuscript version in any

repository, provided it is only made publicly

available 12 months after official publication

or later and provided acknowledgement is

given to the original source of publication

and a link is inserted to the published article

on Springer's website. The link must be

accompanied by the following text: "The final

publication is available at link.springer.com”.

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Surfactin from Bacillus subtilis displays an unexpectedanti-Legionella activity

Clémence Loiseau & Margot Schlusselhuber &

Renaud Bigot & Joanne Bertaux & Jean-Marc Berjeaud &

Julien Verdon

Received: 29 August 2014 /Revised: 29 October 2014 /Accepted: 10 December 2014# Springer-Verlag Berlin Heidelberg 2015

Abstract A contaminant bacterial strain was found to exhibitan antagonistic activity against Legionella pneumophila, thecausative agent of Legionnaires’ disease. The bacterial strainwas identified as a Bacillus subtilis and named B. subtilisAM1. PCR analysis revealed the presence of the sfp gene,involved in the biosynthesis of surfactin, a lipopeptide withversatile bioactive properties. The bioactive substances wereextracted from AM1 cell-free supernatant with ethyl acetateand purified using reversed phase HPLC (RP-HPLC).Subsequent ESI-MS analyses indicated the presence of twoactive substances with protonated molecular ions at m/z 1008and 1036 Da, corresponding to surfactin isoforms. Structuresof lipopeptides were further determined by tandem massspectrometry and compared to the spectra of a commerciallyavailable surfactin mixture. Surfactin displays an antibacterialspectrum almost restricted to the Legionella genus (MICsrange 1–4 μg/mL) and also exhibits a weak activity towardthe amoeba Acanthamoeba castellanii, known to be the natu-ral reservoir of L. pneumophila. Anti-biofilm assays demon-strated that 66 μg/mL of surfactin successfully eliminated90 % of a 6-day-old biofilm. In conclusion, this study reveals

for the first time the potent activity of surfactin againstLegionella sp. and preformed biofilms thus providing newdirections toward the use and the development of lipopeptidesfor the control of Legionella spread in the environment.

Keywords Lipopeptides . Biosurfactants . Surfactin .

Legionella .Bacillus . Biofilms

Introduction

Legionella pneumophila is a Gram-negative opportunisticintracellular human pathogen responsible for Legionnaires’disease, a severe form of pneumonia strongly associated withhuman-made aquatic environments (McDade et al. 1977). Inparticular, many disease outbreaks are linked to air-conditioning cooling towers and evaporative condensers,which can produce contaminated water droplets that are in-haled by passerby (Newton et al. 2010). Within freshwaterenvironments, L. pneumophila bacteria are ubiquitous organ-isms mostly found as parasites of various free-living protozoasuch as amoebae, their natural hosts (Steinert et al. 2002).Free-living amoebae are not solely responsible forL. pneumophila spreading, but they also are considered asbiological water bypasses as they protect intracellular bacteriafrom adverse conditions or biocide treatments (Loret andGreub 2010). Also, upon transfer from natural freshwaterhabitats into anthropogenic systems, L. pneumophila colo-nizes existing complex biofilms and proliferates (Rogerset al. 1994). In biofilms, the microorganisms are irreversiblyassociated with a surface and enclosed in a matrix of extracel-lular polymeric substances of their own origin (Hall-Stoodleyet al. 2004). One of the main advantages of biofilm lifestyle

Clémence Loiseau and Margot Schlusselhuber contributed equally to thiswork.

Electronic supplementary material The online version of this article(doi:10.1007/s00253-014-6317-z) contains supplementary material,which is available to authorized users.

C. Loiseau :M. Schlusselhuber :R. Bigot : J.<M. Berjeaud :J. Verdon (*)Equipe Microbiologie de l’Eau, Université de Poitiers,Ecologie & Biologie des Interactions, UMR CNRS 7267,1 Rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9, Francee-mail: julien.verdon@univ-poitiers.fr

J. BertauxEquipe Ecologie Evolution Symbiose, Université de Poitiers,Ecologie & Biologie des Interactions, UMR CNRS 7267,1 Rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9, France

Appl Microbiol BiotechnolDOI 10.1007/s00253-014-6317-z

Author's personal copy

for L. pneumophila is the increased resistance to stressfulenvironmental conditions and to biocides (Borella et al.2005; Kim et al. 2002). In order to restrain L. pneumophilagrowth, various treatments are currently used (e.g., chlorine,monochloramine, heat shocks) in water systems. However,these treatments are not fully efficient since after a lag periodfollowing the treatment, L. pneumophila is able to rapidly re-colonize the system (Cooper and Hanlon 2010; Thomas et al.2004). Indeed, the resistance afforded to L. pneumophilaagainst harsh conditions by the formation of biofilms and bythe presence of free-living amoebae has implications for thedelivery of drinking water (Dupuy et al. 2011; Wingender andFlemming 2011). Therefore, it is of primary importance tofind new natural antibacterial agents to controlL. pneumophila spread.

Among the diverse array of available biomolecules ofecological interest, lipopeptides (LPs), a class of microbialsecondary metabolites, have received considerable attentionfor their versatile bioactive properties. Indeed, they form themost widely reported class of biosurfactants having lytic andgrowth-inhibitory activities against a broad range of microor-ganisms including bacteria, viruses, and fungi (for reviews,see Ongena and Jacques 2008; Raaijmakers et al. 2010).Among LP producers, Bacillus strains remain the most com-monly used and well-studied organisms. LPs from Bacillusshowed a remarkable structural heterogeneity and are classi-fied in three main families: surfactins, iturins, and fengycins(Ongena and Jacques 2008). Surfactin, that is produced byvarious strains of Bacillus subtilis, is the first LP described inthe literature (Arima et al. 1968) and remains the best knownbiosurfactant. Members of the surfactin family are constitutedof a heptapeptide moiety linked to a β-hydroxylated fatty acidto form a cyclic lactone ring (Kakinuma et al. 1969a, b).Contrarily to iturins and fengycins which exhibit mainly anti-fungal activity, surfactins display antiviral and antibacterialactivities (Ongena and Jacques 2008).

In the present study, we report the identification of aB. subtilis strain with the capacity to inhibit the growth ofthe waterborne opportunistic pathogen L. pneumophila.Biomolecules responsible for the antagonistic activity werepurified and characterized. Their antimicrobial activities weredetermined against selected Gram-positive and Gram-negative indicator strains and the protozoa Acanthamoebacastellanii. Finally, their ability to disturb L. pneumophilaadhesion and mature biofilms was investigated.

Materials and methods

Strains, culture conditions, and reagents

The bacterial strains used in this study are listed in Tables 1and 2. Legionella strains were cultured at 37 °C either on

Table 1 Minimum inhibitory concentration of surfactin againstbacteria of the Legionella genus

Legionella strains MIC (μg/mL)

Legionella bozemanii ATCC 33217 1.5

Legionella dumofii ATCC 33279 1.5

Legionella longbeachae ATCC 33484 1.5

Legionella oakridgensis ATCC 33761 3

Legionella feeleii ATCC 35072 1

Legionella micdadei ATCC 33218 3

Legionella pneumophila Corby (Sg 1) 2

Legionella pneumophila ATCC 33215 (Sg 6) 3

Legionella pneumophila ATCC 33155 (Sg 3) 4

Legionella pneumophila Lens CIP 108286 (Sg 1) 4

Legionella pneumophila ATCC BAA-74 130b (Sg 1) 3

Legionella pneumophila ATCC 33216 (Sg 5) 3

Bacteria (106 CFU/mL) were incubated in BYE medium with twofolddilutions of a 265-μg/mL surfactin stock solution. Results correspond tothe MIC after incubation for 96 h at 37 °C and are the mean of threeindependent experiments. Legionella strains were obtained from variousculture collections: American Type Culture Collection (ATCC) or Col-lection Institut Pasteur (CIP). The Corby strain was kindly provided bythe Centre National de Référence des Légionelles (Lyon, France)

Sg serogroup

Table 2 Minimum inhibitory concentration of surfactin againstselected bacterial strains

Bacterial strains MIC(μg/mL)

Gram-positive Enterococcus faecalis V583 >256

Listeria monocytogenes EGDe ATCCBAA-679

>256

Staphylococcus aureus ATCC 29213 >256

Listeria ivanovii Li4pVS2 >256

Bacillus subtilis LMG 28342 >256

Gram-negative Klebsiella pneumoniae 0502083 >256

Proteus mirabilis ATCC 35659 >256

Enterobacter cloacae DO3 >256

Pseudomonas aeruginosa PA14 >256

Pseudomonas aeruginosa LMG 1242 >256

Aeromonas hydrophila LMG 2844 >256

Salmonella enterica J18 >256

Flavobacterium breve LMG 4011 >256

Escherichia coli LMG 2092 >256

Pseudomonas syringae pv tomato DC3000 >256

Bacteria (106 CFU/mL) were incubated in BHI medium with twofolddilutions of a 265-μg/mL surfactin stock solution. Results correspond tothe MIC after incubation for 24 h at 30 or 37 °C depending on the testedstrain. Results are the mean of three independent experiments. Bacterialstrains were obtained from various culture collections: Library of Micro-biology (LMG, Universiteit Gent, Belgium), American Type CultureCollection (ATCC), Collection Institut Pasteur (CIP). Other bacterialstrains were from the laboratory culture collection

Appl Microbiol Biotechnol

Author's personal copy

buffered charcoal yeast extract (BCYE) agar plates for 96 h orin buffered yeast extract (BYE) liquid medium for 30 h undershaking (150 rpm). L. pneumophila Lens (CIP 108286)(Cazalet et al. 2008) was used as model for biofilm formation.This strain was grown in a supplemented biofilm broth (SBB)as described by Pecastaings et al. (2010). Non-Legionellabacteria were grown for 24 to 48 h either on brain-heartinfusion (BHI) agar plates or broth, at 30 or 37 °C dependingon the tested strain. Biosurfactant production was achieved bycultivating B. subtilis AM1 in mineral salt medium (MSM)supplemented with glucose as the sole carbon source (Janeket al. 2010). A 200-mL working volume was inoculated at anOD600=0.01 from an overnight preculture and incubated on arotary shaker (180 rpm) for 96 h at 17 °C. Growth of the strainAM1 was monitored by measuring the absorbance of theculture at 600 nm. A. castellanii ATCC 30234 was obtainedfrom the American Type Culture Collection. Axenic culturesof Acanthamoebae were grown in peptone yeast glucose(PYG) medium (Schuster 2002) and incubated at 30 °C.Surfactin was purchased from Sigma-Aldrich (St. Louis,MO, USA). All other reagents were purchased from Sigma-Aldrich (Saint-Louis, MO, USA) unless stated otherwise.

Identification of bacterial strain

Strain AM1was identified by sequencing of its 16S ribosomalDNA (rDNA) gene. Genomic DNAwas isolated from a 1-dayliquid culture using a Wizard SV Genomic DNA purificationkit (Promega, Madison, WI, USA) according to the manufac-turer’s instructions and used as a template for polymerasechain reaction (PCR) amplification using primers 515F (5′-GTG CCA GCM GCC GCG GTA A-3′) and 1492R (5′-GGTTAC CTT GTT ACG ACT T-3′). Amplification was carriedout as follows: denaturation at 94 °C for 30 s, annealing at50 °C for 30 s, and extension at 72 °C for 1 min, for a total of30 cycles, followed by a final elongation at 72 °C for 5 min.Then, the PCR products were subjected to 1 % agarose gelelectrophoresis and further purified using a NucleoSpin® Geland PCR Clean-up kit (Macherey-Nagel, Bethlehem, PA,USA). DNA sequencing was completed with the ABI PrismBigDye terminator v3.1 sequencing kit (Applied Biosystems,Carlsbad, CA, USA) and then analyzed by an automatic DNAsequencer, ABI Prism 3730 genetic analyzer (AppliedBiosystems, Carlsbad, CA, USA). The 830-bp sequencesobtained with the 1492R primer was compared to rDNAsequences obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). The partial 16S rDNAnucleotide sequence of B. subtilis AM1 has been submittedto the GenBank database under accession number KJ778029.Moreover, the strain AM1 has been deposited in the BCCM/LMG bacteria collection under accession number LMG28342.

Detection of the sfp gene by PCR

The sfp 675-bp fragment (Nakano et al. 1992), correspondingto the B. subtilis sfp gene (GenBank accession numberX63158.1) at positions 167–841, was PCR-amplified usingtwo oligonucleotide primers of sfp-f (5′-ATG AAG ATT TACGGA ATT TA-3′) and sfp-r (5′-TTA TAA AAG CTC TTCGTA CG-3′) as described elsewhere (Hsieh et al. 2004). Thepartial sfp gene nucleotide sequence of B. subtilis has beensubmitted to the GenBank database under accession numberKJ778030.

Extraction and purification of biosurfactants

B. subtilisAM1was grown inMSMmedium for 96 h at 17 °C.Bacteria were removed by centrifugation (10,000×g, 30 min,4 °C), and the resulting supernatant was sterilized by filtrationthrough a 0.22-μm syringe filter (Millipore, Germany). Thecell-free supernatant was extracted three times with ethyl ace-tate (1:1, v/v), and the collected organic fractions were evapo-rated under vacuum. The crude biosurfactants were dissolved in5 mL of H2O/acetonitrile (ACN) (3:2, v/v) mixture and sepa-rated by RP-HPLC. HPLC purification was conducted on aChromolith® SpeedROD RP-18e reverse-phase HPLC column(4.6×50 mm) (Merck Millipore, Billerica, MA, USA) with aDionex P680 HPLC pump, fitted with a Dionex Ultimate 3000detector. Elution was monitored at 205 and 214 nm. Separationwas carried out usingH2O/ACN/formic acid 0.2% (v/v) solventsystem. After an initial 2-min wash with 60 % ACN, elutionwas achieved in 23min at a flow rate of 0.8mL/minwith an 18-min linear gradient from 60 to 100 % ACN, followed by a 5-min wash with 100 % ACN. Quantification of biosurfactantswas performed using the commercial solution as a standard.Each peak area was calculated after purification of the com-mercial surfactin solution at 214 nm for the evaluation of thepeptidicmoiety. All the collected fractions were lyophilized andstored at −20 °C for further studies.

Mass spectrometry analyses

The molecular masses of biosurfactants were determined byelectrospray ionization mass spectrometry (ESI-MS) with aXevo Q-TOF (Waters, Milford, MA, USA) mass spectrome-ter. Samples were suspended in 50 % ACN/0.2 % formic acid(v/v) and analyzed, in positive mode, by infusion at a flow rateof 0.5 mL/min. The spray voltage was set to 3.0 kV, the sourcetemperature to 120 °C, and the desolvation temperature to250 °C. LC MS/MS mass spectra were performed in the MSE

mode (Waters,Milford,MA, USA). Briefly, in theMSEmode,mass spectrometric scans alternate all along the experimentbetween low (10 V) and high (ramping from 30 to 60 V)fragmentation energy delivering for the same LC separation(isocratic elution for 5 min in ACN containing 0.2 % formic

Appl Microbiol Biotechnol

Author's personal copy

acid) of two chromatograms corresponding to MS and MS/MS analyses respectively.

Spot on lawn assay

The target strain, L. pneumophila Lens, was spread (100μL ofa suspension adjusted to 108 CFU/mL) onto a BCYE agarplate. Then, 10 μL of an overnight culture of strain AM1 wasspotted onto the surface of the agar plate before incubation for96 h at 37 °C.

Determination of the minimal inhibitory concentrationof surfactin

The minimal inhibitory concentration (MIC) of surfactin wasmeasured according to the method detailed by Verdon et al.(2008). MIC was defined as the lowest concentration ofsurfactin that totally inhibits the visible growth of a selectedstrain after a chosen incubation period.

Determination of bactericidal effect

The bactericidal effect of surfactin on L. pneumophila Lens inexponential-phase was determined as previously described(Verdon et al. 2008).

Viability of amoebae in the presence of surfactin

The effects of surfactin on membrane integrity of amoebatrophozoites were assessed by monitoring the fluorescenceof the DNA-intercalating dye SYTOX Green (Invitrogen,Carlsbad, CA, USA) in cells with compromised membranes.Briefly, A. castellanii ATCC 30234 was cultured axenically at30 °C and split the day prior such that 2.104 trophozoites werepresent in each well of a 96-well black plate. Serial dilutionsof surfactin were done in PAS buffer (Page’s modified Neff’samoeba saline) pH 6.5 containing 2 μMof the fluorescent dyeSYTOX Green. A. castellanii was treated in a total volume of100 μL for 2 h at 30 °C. For spontaneous lysis of the amoeba(0 % value), incubation was done in buffer and for maximumpermeabilization (100 % value), and 0.1 % Triton X-100 wasadded to the buffer. After incubation, fluorescence of thesamples was measured at 535 nm after excitation at 488 nmwith a spectrofluorometer (Tristar2 LB 942 MultimodeReader, Berthold Technologies). Experiments were done induplicate and data are expressed as mean±standard deviation.Results were evaluated statistically using a one-way analysisof variance (ANOVA) with a Bonferroni correction posttestusing GraphPad Prism version 5.04 for Windows (GraphPadSoftware, San Diego, CA, USA).

Biofilm production

Single-species biofilms were grown in the Calgary BiofilmDevice (CBD;MBECAssay™ P&G, Innovotech, Edmonton,AB, Canada) as previously described by Ceri et al. (1999)with some modifications. The CBD were coated overnightwith 200 μL per well of SBB medium prior inoculation with150 μL of L. pneumophila Lens suspension (106 CFU/mL)per well. Sealed microtiter plates were incubated for 6 days at37 °C under agitation (110 rpm). During the course of incu-bation, the medium was renewed after 3 days.

Effect of surfactin on mature biofilms

A 6-day-old L. pneumophila biofilm was challenged withincreasing concentrations of surfactin. After a gentle rinsingfor 1 min in saline buffer, 96-well plate lids, with biofilmsattached to the pegs, were deposited in 96-well plate contain-ing 200 μL per well of SBB medium supplemented withvarious surfactin concentrations. Then, the challenging micro-titer plate was incubated for 2 h at 37 °C under gentle agitation(110 rpm). Afterward, the lid was deposited in a new 96-wellplate containing 200 μL of BYE per well. The plate wassonicated in a bath sonicator during 5 min at 100% ultrasoundpower (Bioblock Scientific 86480, Fisher, Illkirch, France).Cell quantificationwas made byCFU counting on BCYE agarplates. Experiments were performed, at least, in two internalreplicates and three independent replicates. Results were eval-uated statistically as described above.

Effect of surfactin on Legionella adhesion

To assess the effect of surfactin on Legionella adhesion, a sub-MIC concentration of surfactin was added to the liquid medi-um at a final concentration of 2μg/mL during the coating step.After 6 days of contact, the effect of surfactin on cells adhe-sion was analyzed by CFU counting.

Microscopic observation of L. pneumophila biofilm

For microscopic studies, DsRed-labeled L. pneumophila Lens(Bigot et al. 2013) biofilms were grown in glass bottom 24-well microtiter plates. The surface was coated overnight withSBB medium prior inoculation of the wells with 2.106 CFU/mL from a fresh culture. Biofilms were grown under staticconditions at 37 °C with medium renewal after 3 days ofincubation. Afterward, 6-day-old biofilms were treated for2 h with 66 μg/mL of surfactin. The supernatant was thenreplaced by fresh SBB medium. Biofilms were visualizedusing an inverted epifluorescent microscope (Axio ObserverA1, Zeiss) with an ApoTomemodule (structured illumination)equipped with a×63/1.25 objective (oil immersion). Pictureswere taken using the AxioVision 4.8.1 software and processed

Appl Microbiol Biotechnol

Author's personal copy

with the software Imasis7.4.1 and Zen (Zeiss). Each picture isrepresentative of the results of, at least, two independentassays.

Results

Identification of B. subtilis AM1

A contaminant strain was found to display an anti-Legionellaactivity as shown by direct antagonism on agar plate (Fig. 1).Besides the anti-Legionella activity, the bacterial strain alsodisplayed a hemolytic activity, as inoculating blood sheep agarplates with this strain resulted in clear rings of lysed erythro-cytes around colonies (data not shown). Comparison of an830-bp 16S rDNA nucleotide sequence from the isolatedstrain with sequences in the GenBank database showed99 % identity with the corresponding sequences fromB. subtilis strain RC22, B. subtilis strain F3-2, and Bacilluslicheniformis strain M2-3. Subsequently, to confirm the iden-tification, sequencing of the sfp gene was performed. The sfpgene, encoding an enzyme belonging to the superfamily of 4′-phosphopantetheinyl transferase, was required for productionof surfactin (Hsieh et al. 2004), a widely produced hemolyticlipopeptide by members of the genus Bacillus and moreparticularly B. subtilis (Peypoux et al. 1999). The two specificprimers (sfp-f and sfp-r) allowed amplification of the expected675-bp fragment indicating that the contaminant strain has thecapacity to produce surfactin (Fig. S1). Sequence of 675-bpfragment showed the highest identity score of 99 % with thesfp gene of B. subtilis A21 (GenBank accession numberJN005771). Taken together, our results indicated that the

contaminant strain was a B. subtilis, so it was namedB. subtilis AM1 and recorded as B. subtilis LMG 28342.

Purification and characterization of the anti-Legionellabiosurfactants

Biosurfactants produced by B. subtilis AM1 were isolatedusing a two-step purification method. First, biosurfactantswere partially purified from the culture supernatant by ethylacetate extraction and were further purified using reverse-phase HPLC on a C18 column. The crude extract obtainedafter extraction was resolved into seven fractions as shownin Fig. 2a. The collected fractions were tested for their anti-Legionella activity, and only two fractions, corresponding topeaks at 11.2 and 15.3 minutes, were found to be activeagainst L. pneumophila Lens (Table S1). Hereafter, bothactive fractions were characterized by ESI-MS. For the firstfraction, molecular ions were observed at m/z 1008.62,1030.64, and 1046.57 (Fig. S2) which were assigned tothe protonated molecular ion, the sodium (22 Da higher)and potassium (38 Da higher) adducts, respectively. For thesecond fraction, molecular ions were observed at m/z1036.63, 1058.62, and 1074.59 (Fig. S2) which were alsoassigned to the protonated molecular ion and the sodium andpotassium adducts, respectively. The two intense pseudo-molecular ions [M + H]+ at m/z 1008 and 1036 wereconsistent with the presence of surfactin (Kowall et al.1998). The main product ions obtained are displayed inFig. 2b. A LC-MS/MS analysis was conducted on the activefractions. The fragmentation spectrum obtained for the firstfraction was dominated by an ion peak at m/z 685 whichwas attributed to the product ion [NH2-AA2-AA7-COOH +H]+ (Fig. 2c). Moreover, the ion peak at m/z 895 indicatedthe loss of 113 Da, which derived from a double cleavage ofthe ester bond of the cyclic skeleton and of the peptidicbond between the last two C-terminal amino acid residues.So, the presence of this ion was used to identify the leucineor isoleucine residue in position 7 (113 Da) (Fig. 2c). TheMS/MS spectrum of parent ion 1036 gave similar resultsand allowed us to identify a fatty acid chain length of 15carbons for the lipid moeity (Fig. 2b). The two anti-Legionella biosurfactants produced by B. subtilis AM1 wereassigned to surfactin isoforms both bearing a leucine or anisoleucine residue at position 7. However, in order to con-firm this hypothesis, we analyzed a commercial solution ofsurfactin to identify all the present isoforms and to checktheir anti-Legionella activity. The same protocol as describedabove was applied to the commercial surfactin. The crudeextract was resolved into 12 fractions (Fig. S3), amongwhich five, corresponding to peaks with retention times of10.1, 11.8, 14.2, 15.3, and 17.4 min, were found active(Table S1). All the active fractions were then further char-acterized by ESI-MS and LC-MS/MS. Pseudo-molecular

Fig. 1 Spot on lawn assay of B. subtilis AM1 (central colony) againstL. pneumophila Lens

Appl Microbiol Biotechnol

Author's personal copy

ions and main product ions observed on the mass spectra ofsurfactin isomers are displayed in Fig. S3. Thus, tensurfactin isoforms, including the two forms produced byB. subtilis AM1, were characterized and differed by fattyacid chain length as well as the amino acid residue in theseventh position which is consistent with the literature(Kowall et al. 1998). It has to be noted that we detectedmainly isoforms bearing a leucine residue in position 7. Inthe tested fractions, isoforms with a valine residue in posi-tion 7 represent only from 5 to 16 % of the lipopeptidesaccording to the comparison of peaks areas. As all testedisolated fractions were active against L. pneumophila Lens,and because large amounts of active compounds were re-quired, we decided to realize the subsequent biological testswith the commercial surfactin solution.

Antibacterial spectrum of surfactin

In order to determine the putative sensitivity of various bac-terial strains, including different species of Legionella, tosurfactin, an antibacterial activity assay was performed and

the minimum inhibitory concentration (MIC) was determinedfor each tested strain. The results are given in Table 1 forLegionella strains and in Table 2 for non-Legionella strains.Surfactin was found active against all the Legionella strainstested, including non-pneumophila, pneumophila serogroup1, and pneumophila non-serogroup 1 strains (Table 1).Moreover, the LPs inhibited the growth of all the tested strainsof L. pneumophila at low concentrations (2–4μg/mL). Similarresults were obtained for other species of Legionella, such asL. bozemanii, L. dumofii, L. longbeachae, L. oakridgensis,L. feeleii, and L. micdadei, with MICs ranged from 1 to 4 μg/mL (Table 1). In contrast, all the other bacterial strains testedwere resistant to surfactin, even at the highest tested concen-tration (265 μg/mL) (Table 2). Surfactin also displayed bac-tericidal effect against exponential phase L. pneumophila, as atreatment for 1 h with 8 μg/mL surfactin led to a bacterialpopulation reduction of 2 log (data not shown). Taken togeth-er, these results indicated that surfactin is active, in vitro, atvery low concentrations against Legionella sp., displays anantibacterial spectrum almost restricted to this genus and has abactericidal potential.

Fig. 2 Purification andcharacterization of biosurfactantsproduced by B. subtilis AM1. aReverse-phase HPLC elutionprofile of biosurfactants producedby B. subtilis AM1. Detectionwas carried out by measuring theabsorbance at 205 nm (gray line)and 214 nm (black line). Activefractions against L. pneumophilawere indicated by arrows. Elutiongradient, expressed in %acetonitrile, is represented by adashed line. b Pseudo-molecularions and main product ionsobtained in positive mode fromESI-MSn (n=1, 2) analyses ofpurified active HPLC fractionsfrom ethyl acetate extract ofB. subtilis AM1 cell-freesupernatant. c Determinedstructure of purified surfactin(1007 Da) based on massspectrometric analyses

Appl Microbiol Biotechnol

Author's personal copy

Effect of surfactin on amoebae viability

As several protozoa, such as free-living amoebae, feed onbiofilms leading to L. pneumophila phagocytosis, and subse-quently proliferation, the sensitivity of A. castellanii was alsoevaluated in the presence of various concentrations ofsurfactin. SYTOX Green, a high-affinity nucleic acid stainthat easily penetrates cells with compromised plasma mem-branes, was used to assess the permeability of A. castellanii(Fig. 3). The results show that SYTOX Green penetratedtreated cells, suggesting permeabilization of the membrane.Furthermore, the number of permeabilized cells increasedwith surfactin concentrations. The highest surfactin concen-tration used (66 μg/mL) induced almost 30 % perme-abilization. Therefore, results indicated that surfactin is ableto permeabilize trophozoites of A. castellanii but at highconcentrations.

Effect of surfactin on Legionella adhesion and biofilmpersistence

Biofilms were grown over 6 days on MBEC™-P&G polysty-rene pegs surface. The disruption of preformed biofilm afterincubation for 2 h with surfactin mixture is presented in Fig. 4.Compared to the untreated control, no biofilm detachmentwas observed with surfactin concentrations ranging from 1to 16.5 μg/mL. At concentrations of 33 and 66 μg/mL,surfactin significantly reduced the amount of biofilm attachedon pegs by 70 % (p<0.01) and 90 % (p<0.001), respectively(Fig. 4). However, the preconditioning of the surface followedby a continuous treatment with 2 μg/mL surfactin over the 6-day-period of incubation was not sufficient to avoid the initialattachment of planktonic L. pneumophila cells and the

maturation of the biofilm, as no differences were observed incomparison with the control (data not shown).

Microscopic observation of the anti-biofilm activityof surfactin

In order to visualize effects of surfactin on L. pneumophilapreformed biofilm, DsRed labeled L. pneumophila Lens wereused to form 6-day-old biofilms on glass-bottom 24-wellmicrotiter plates under static conditions. The disruption ofpreformed biofilm after 2 h contact with 66μg/mL of surfactinor its diluent was analyzed using an epifluorescent microscopeequipped with an ApoTome module for a structured illumina-tion. The untreated biofilm appeared thick (30 μm) and pre-sented densely packed bacteria (Fig. 5a) while the 2-h treatedbiofilm was thinner, between 6 and 14 μm, with few attachedcells that appeared dispersed on the glass surface (Fig. 5b). Atlow magnification (×20 lens), large and numerous aggregatesin the L. pneumophila biofilm on the glass surface wereobserved in the control, while very few were visible in thetreated sample (data not shown). In conclusion, a 2-h treat-ment with 66 μg/mL surfactin successfully disrupt the major-ity of a 6-day-old L. pneumophila biofilm, which was consis-tent with the results of CFU counting obtained in the bacterialadhesion assay.

Discussion

B. subtilis strains are known to produce a wide variety ofstructurally diverse secondary metabolites with great potentialfor biotechnological, biocontrol, and therapeutic applications

0

10

20

30

40

50

60

70

80

90

100

0 2 4.1 8.2 16.5 33 66

Cel

l per

mea

biliz

atio

n(%

)

Surfactin concentration (µg/mL)

***

***

Fig. 3 Permeabilization ofAcanthamoeba castellanii in thepresence of surfactin. Amoebaewere treated with surfactinconcentrations ranging from 0 to66 μg/mL. Error bars indicatestandard deviation from twoindependent experiments;statistical significance incomparison to the control isshown as follows: ***p<0.0001

Appl Microbiol Biotechnol

Author's personal copy

(Gudina et al. 2013; Ongena and Jacques 2008; Sen 2010).Among these natural compounds, cyclic lipopeptides of thesurfactin, iturin, and fengycin families have well-recognizedpotential because of their surfactant and antimicrobial proper-ties (Stein 2005). Compilation of recent findings demonstratesspecific roles for such biomolecules beyond their antibioticactivity, hence contributing to the survival of microorganismsin their natural habitat (Raaijmakers et al. 2010). TheB. subtilis AM1 strain described in this study was selectedbecause of its anti-Legionella activity. To date, only oneBacillus strain was shown to exhibit a potent activity towardL. pneumophila (Temmerman et al. 2007). However, the

molecules responsible for the antagonistic activity were notcharacterized. Because the strain AM1 displayed a hemolyticactivity when grown on blood agar plates, we hypothesizedthat the bacteria produced biosurfactants during its growth, aspreviously shown (Hsieh et al. 2004). Using a linear gradientof acetonitrile and water, the crude extract was resolved byRP-HPLC into two active fractions. Mass spectra analysis ofcollected active fractions indicated that these fractionscorresponded to surfactin isoforms with molecular masses of1007 and 1035 Da, respectively. The difference of 28 Dasuggested that they only varied by two carbons in the fattyacid chain and/or could present differences in their amino acid

0

20

40

60

80

100

120

0 4.1 8.2 16.5 33 66

Atta

ched

bio

film

(%)

Surfactin concentration (µg/ml)

**

***

Fig. 4 RemainingL. pneumophila attached cells onpolystyrene pegs surface in thepresence of surfactin. Biofilmswere treated 2 h with surfactinconcentrations ranging from 0 to66 μg/mL. Experiments weredone in triplicate. Error barsindicate standard deviation fromthree independent experiments;statistical significance incomparison to the control isshown as follows: **p<0.01;***p<0.0001

Fig. 5 Apotome imaging of 6-day-old biofilms formed byDsRed-labeled L. pneumophila.Biofilms were treated 2 h eitherwith a ethanol as control or b66-μg/mL surfactin. Each pictureis representative of the results ofat least two independentexperiments

Appl Microbiol Biotechnol

Author's personal copy

composition (Tang et al. 2010). The loss of 113 Da in the MS/MS analyses confirmed that the surfactins produced by AM1possessed the same peptide sequence, with a Leucine orIsoleucine but not Valine residue in position 7, and differedby the fatty acid chain length. In order to confirm that theAM1-produced LPs are effectively surfactin isoforms, wecompared their mass spectra, MS, and MS/MS, with thoseof a commercially purchased surfactin solution. As previouslydescribed (Bais et al. 2004), the solution was found to becomposed of a mixture of surfactin isoforms. Indeed, tenisoforms were identified including the two produced byB. subtilis AM1. More importantly, all the purified surfactinisoforms, whatever their origin, were found to be activeagainst L. pneumophila, the causative agent of Legionnaires’disease. Surfactins with such structures and produced byvarious B. subtilis strains were previously reported in theliterature (Abdel-Mawgoud et al. 2008; Geetha et al. 2010;Haddad et al. 2008; Peypoux et al. 1994). To date, only fewstudies focused on the antagonistic activity of surfactin towardhuman pathogenic bacteria (Huang et al. 2007; Hwang et al.2007; Sabate and Audisio 2013). It is not surprising as hemo-lytic activity of most of the reported biosurfactants includingsurfactin and the lack of clinical data on the use and validationof such molecules in animal models and human volunteerspose, until now, a major problem for therapeutic applications.

MIC determination revealed that surfactin displayed anantibacterial activity spectrum restricted to the Legionellagenus (1–4 μg/mL). Indeed, none of the other tested strainswas sensitive, even at 265 μg/mL. According to the literatureon the antibacterial activities of purified or commerciallypurchased standard surfactins, few susceptible species tosurfactin were reported. Indeed, antibacterial activity wasshown against Escherichia coli (Huang et al. 2008),Mycoplasma spp. (Fassi Fehri et al. 2007; Nir-Paz et al.2002; Nissen et al. 1997; Vollenbroich et al. 1997),Pseudomonas syringae (Bais et al. 2004), Paenibacilluslarvae (Sabate et al. 2009), Listeria monocytogenes (Sabateand Audisio 2013), Salmonella enteritidis (Huang et al. 2011),Vibrio spp. (Xu et al. 2014), and Xanthomonas arboricola(Dimkic et al. 2013). When determined by microtiter plateassays, MIC values ranged from 6.25 to 156 μg/mL.Therefore, some of our results are in contradiction with theliterature. These discrepancies could be explained by the useof different strains belonging to same species, by differentexperimental conditions, or by the use of different surfactinisoforms. In the particular case of P. syringae DC3000, thestrain was previously shown to be sensitive to surfactin (Baiset al. 2004). In the latter study, the authors found that surfactinexhibits, in vitro, a MIC against P. syringae of approximately25 μg/mL. However, it has to be noted that the MIC determi-nation assay was performed at 37 °C, while we determined theMIC against P. syringae DC3000 at 28 °C, the routinely usedgrowth temperature for this microorganism (Uppalapati et al.

2007; Zhao et al. 2003). Taken together, these results suggestthat surfactin is very efficient in inhibiting Legionella sp. ascompared to other bacterial genera.

L. pneumophila is ubiquitously found in freshwater andcould survive within biofilms and free-living amoebae(Declerck 2010). Several protozoa, such as free-living amoe-bae, feed on biofilm leading to bacterial phagocytosis.However, L. pneumophila has the ability to resist phagocytosisandmultiply within amoebae (Escoll et al. 2013). Therefore, wewere interested in studying the effects of surfactin on amoebaeand biofilms formed by Legionella. Protozoa were previouslyreported to be sensitive to various bacterial LPs, providingprotection against protozoan predation in soil ecosystems(Mazzola et al. 2009).We showed that surfactin is poorly activeagainst A. castellanii, suggesting that this LP couldpermeabilize amoeba at high concentrations, leading to a slightdecrease of cell viability. Nevertheless, LPs produced by vari-ous bacterial genera exhibited considerable structural diversity,including differences in the number, nature, and configuration(L or D) of the amino acids in the macrocyclic peptide ring, aswell as in the type and length of the fatty acid tail (Ongena andJacques 2008; Peypoux et al. 1999; Raaijmakers et al. 2006).How these differences in structural features affect the activityagainst protozoan grazing or determine the spectrum of targetprotozoan species remains to be addressed.

For Bacillus sp. and Pseudomonas sp., LPs play an impor-tant role in surface attachment and biofilm formation forproducers, although the outcome may differ depending onthe type of LP (Raaijmakers et al. 2010). In addition, LPswere also reported to adversely affect the attachment to sur-faces and biofilm formation by other microorganisms, leadingsometimes to the breakdown of existing biofilms (Kuiper et al.2004; Mireles et al. 2001). In our study, we found thatsurfactin was able to breakdown L. pneumophila preformedbiofilms but did not prevent the biofilm attachment. The anti-preformed biofilm activity was already shown for surfactinagainst Salmonella, but in this case, contrarily to the effect onLegionella biofilms, the LPs displayed a preventive actiontoward the biofilm initial development (Mireles et al. 2001).

In conclusion, the present study describes the identificationof surfactin from the supernatant of a contaminant B. subtilis.Surfactin possesses a strong anti-Legionella activity whateverthe serogroup or the species tested. The low displayed MICand the sensitivity of all Legionella tested make surfactin avery promising candidate for a novel-specific drug againstLegionella. However, its hemolytic potential is a barrier thatdeserves to be by-passed using structure/function relationshipstudies, as previously shown (Symmank et al. 2002). Surfactinis also able to breakdown existing biofilms of L. pneumophilasuggesting that it could represent a potent tool for the biolog-ical control of the pathogen in water treatment industry, al-though additional experiments are needed to demonstrate howeffective this antagonist would be under field conditions.

Appl Microbiol Biotechnol

Author's personal copy

Acknowledgments The authors would like to thank Daniel Guyonnet(University of Poitiers) for help in DNA sequencing. Clemence Loiseau issupported by a grant from the French Minister of Research.

References

Abdel-Mawgoud AM, Aboulwafa MM, Hassouna NA (2008)Characterization of surfactin produced by Bacillus subtilis isolateBS5. Appl Biochem Biotechnol 150(3):289–303. doi:10.1007/s12010-008-8153-z

Arima K, Kakinuma A, Tamura G (1968) Surfactin, a crystallinepeptidelipid surfactant produced by Bacillus subtilis: isolation, char-acterization and its inhibition of fibrin clot formation. BiochemBiophys Res Commun 31(3):488–494

Bais HP, Fall R, Vivanco JM (2004) Biocontrol of Bacillus subtilisagainst infection of Arabidopsis roots by Pseudomonas syringae isfacilitated by biofilm formation and surfactin production. PlantPhysiol 134(1):307–319. doi:10.1104/pp. 103.028712

Bigot R, Bertaux J, Frere J, Berjeaud JM (2013) Intra-amoeba multipli-cation induces chemotaxis and biofilm colonization and formationfor Legionella. PLoS One 8(10):e77875. doi:10.1371/journal.pone.0077875

Borella P, Guerrieri E, Marchesi I, Bondi M, Messi P (2005) Waterecology of Legionella and protozoan: environmental and publichealth perspectives. Biotechnol Annu Rev 11:355–380. doi:10.1016/S1387-2656(05)11011-4

Cazalet C, Jarraud S, Ghavi-Helm Y, Kunst F, Glaser P, Etienne J,Buchrieser C (2008) Multigenome analysis identifies a worldwidedistributed epidemic Legionella pneumophila clone that emergedwithin a highly diverse species. Genome Res 18(3):431–441. doi:10.1101/gr.7229808

Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A (1999) TheCalgary Biofilm Device: new technology for rapid determination ofantibiotic susceptibilities of bacterial biofilms. J Clin Microbiol37(6):1771–1776

Cooper IR, Hanlon GW (2010) Resistance of Legionella pneumophilaserotype 1 biofilms to chlorine-based disinfection. J Hosp Infect74(2):152–159. doi:10.1016/j.jhin.2009.07.005

Declerck P (2010) Biofilms: the environmental playground of Legionellapneumophila. Environ Microbiol 12(3):557–566. doi:10.1111/j.1462-2920.2009.02025.x

Dimkic I, Zivkovic S, Beric T, Ivanovic Z, Gavrilovic V, Stankovic S,Fira D (2013) Characterization and evaluation of two Bacillusstrains, SS-12.6 and SS-13.1, as potential agents for the control ofphytopathogenic bacteria and fungi. Biol Control 65:312–321

Dupuy M, Mazoua S, Berne F, Bodet C, Garrec N, Herbelin P, Menard-Szczebara F, Oberti S, Rodier MH, Soreau S, Wallet F, Hechard Y(2011) Efficiency of water disinfectants against Legionellapneumophila and Acanthamoeba. Water Res 45(3):1087–1094.doi:10.1016/j.watres.2010.10.025

Escoll P, Rolando M, Gomez-Valero L, Buchrieser C (2013) Fromamoeba to macrophages: exploring the molecular mechanisms ofLegionella pneumophila infection in both hosts. Curr TopMicrobiolImmunol 376:1–34. doi:10.1007/82_2013_351

Fassi Fehri L, Wroblewski H, Blanchard A (2007) Activities of antimi-crobial peptides and synergy with enrofloxacin againstMycoplasmapulmonis. Antimicrob Agents Chemother 51(2):468–474. doi:10.1128/AAC. 01030-06

Geetha I, Manonmani AM, Paily KP (2010) Identification and character-ization of a mosquito pupicidal metabolite of a Bacillus subtilissubsp. subtilis strain. Appl Microbiol Biotechnol 86(6):1737–1744. doi:10.1007/s00253-010-2449-y

Gudina EJ, Rangarajan V, Sen R, Rodrigues LR (2013) Potential thera-peutic applications of biosurfactants. Trends Pharmacol Sci 34(12):667–675. doi:10.1016/j.tips.2013.10.002

Haddad NI, Liu X, Yang S, Mu B (2008) Surfactin isoforms fromBacillus subtilis HSO121: separation and characterization. ProteinPept Lett 15(3):265–269

Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms:from the natural environment to infectious diseases. Nat RevMicrobiol 2(2):95–108. doi:10.1038/nrmicro821

Hsieh FC, Li MC, Lin TC, Kao SS (2004) Rapid detection and charac-terization of surfactin-producing Bacillus subtilis and closely relatedspecies based on PCR. Curr Microbiol 49(3):186–191. doi:10.1007/s00284-004-4314-7

Huang X, Lu Z, Bie X, Lu F, Zhao H, Yang S (2007) Optimization ofinactivation of endospores of Bacillus cereus by antimicrobiallipopeptides from Bacillus subtilis fmbj strains using a responsesurface method. Appl Microbiol Biotechnol 74(2):454–461. doi:10.1007/s00253-006-0674-1

Huang X, Suo J, Cui Y (2011) Optimization of antimicrobial activity ofsurfactin and polylysine against Salmonella enteritidis in milk eval-uated by a response surface methodology. Foodborne Pathog Dis8(3):439–443. doi:10.1089/fpd.2010.0738

Huang X, Wei Z, Zhao G, Gao X, Yang S, Cui Y (2008) Optimization ofsterilization of Escherichia coli in milk by surfactin and fengycinusing a response surface method. Curr Microbiol 56(4):376–381.doi:10.1007/s00284-007-9066-8

Hwang YH, Park BK, Lim JH, Kim MS, Park SC, Hwang MH, Yun HI(2007) Lipopolysaccharide-binding and neutralizing activities ofsurfactin C in experimental models of septic shock. Eur JPharmacol 556(1–3):166–171. doi:10.1016/j.ejphar.2006.10.031

Janek T, Lukaszewicz M, Rezanka T, Krasowska A (2010) Isolation andcharacterization of two new lipopeptide biosurfactants produced byPseudomonas fluorescens BD5 isolated from water from the ArcticArchipelago of Svalbard. Bioresour Technol 101(15):6118–6123.doi:10.1016/j.biortech.2010.02.109

Kakinuma AHM, Isono M, Tamura G, Arima K (1969a) Determinationof amino acid sequence in surfactin, a crystalline peptidolipid sur-factant produced by Bacillus subtilis. Agric Biol Chem 33:971–997

Kakinuma ASH, IsonoM, Tamura G, Arima K (1969b) Determination offatty acid in surfactin and elucidation of the total structure ofsurfactin. Agric Biol Chem 33:973–976

Kim BR, Anderson JE, Mueller SA, Gaines WA, Kendall AM (2002)Literature review—efficacy of various disinfectants againstLegionella in water systems. Water Res 36(18):4433–4444

Kowall M, Vater J, Kluge B, Stein T, Franke P, Ziessow D (1998)Separation and characterization of surfactin isoforms produced byBacillus subtilis OKB 105. J Colloid Interface Sci 204(1):1–8

Kuiper I, Lagendijk EL, Pickford R, Derrick JP, Lamers GE, Thomas-Oates JE, Lugtenberg BJ, Bloemberg GV (2004) Characterization oftwo Pseudomonas putida lipopeptide biosurfactants, putisolvin Iand II, which inhibit biofilm formation and break down existingbiofilms. Mol Microbiol 51(1):97–113

Loret JF, Greub G (2010) Free-living amoebae: biological by-passes inwater treatment. Int J Hyg Environ Health 213(3):167–175. doi:10.1016/j.ijheh.2010.03.004

Mazzola M, de Bruijn I, Cohen MF, Raaijmakers JM (2009) Protozoan-induced regulation of cyclic lipopeptide biosynthesis is an effectivepredation defense mechanism for Pseudomonas fluorescens.Appl Environ Microbiol 75(21):6804–6811. doi:10.1128/AEM. 01272-09

McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR(1977) Legionnaires’ disease: isolation of a bacterium and demon-stration of its role in other respiratory disease. N Engl J Med297(22):1197–1203. doi:10.1056/NEJM197712012972202

Mireles JR 2nd, Toguchi A, Harshey RM (2001) Salmonella entericaserovar Typhimurium swarming mutants with altered biofilm-

Appl Microbiol Biotechnol

Author's personal copy

forming abilities: surfactin inhibits biofilm formation. J Bacteriol183(20):5848–5854. doi:10.1128/JB.183.20.5848-5854.2001

Nakano MM, Corbell N, Besson J, Zuber P (1992) Isolation and charac-terization of sfp: a gene that functions in the production of thelipopeptide biosurfactant, surfactin, in Bacillus subtilis. Mol GenGenet MGG 232(2):313–321

Newton HJ, Ang DK, van Driel IR, Hartland EL (2010) Molecularpathogenesis of infections caused by Legionella pneumophila.Clin Microbiol Rev 23(2):274–298. doi:10.1128/CMR. 00052-09

Nir-Paz R, Prevost MC, Nicolas P, Blanchard A, Wroblewski H (2002)Susceptibilities of Mycoplasma fermentans and Mycoplasmahyorhinis to membrane-active peptides and enrofloxacin in humantissue cell cultures. Antimicrob Agents Chemother 46(5):1218–1225

Nissen E, Pauli G, Vater J, Vollenbroich D (1997) Application of surfactinfor mycoplasma inactivation in virus stocks. Vitro Cell Dev BiolAnim 33(6):414–415. doi:10.1007/s11626-997-0056-8

OngenaM, Jacques P (2008) Bacillus lipopeptides: versatile weapons forplant disease biocontrol. Trends Microbiol 16(3):115–125. doi:10.1016/j.tim.2007.12.009

Pecastaings S, Berge M, Dubourg KM, Roques C (2010) SessileLegionella pneumophila is able to grow on surfaces and generatestructured monospecies biofilms. Biofouling 26(7):809–819. doi:10.1080/08927014.2010.520159

Peypoux F, Bonmatin JM, Labbe H, Grangemard I, Das BC, Ptak M,Wallach J, Michel G (1994) [Ala4]surfactin, a novel isoform fromBacillus subtilis studied by mass and NMR spectroscopies. Eur JBiochem / FEBS 224(1):89–96

Peypoux F, Bonmatin JM, Wallach J (1999) Recent trends in the bio-chemistry of surfactin. Appl Microbiol Biotechnol 51(5):553–563

Raaijmakers JM, de Bruijn I, de Kock MJ (2006) Cyclic lipopeptideproduction by plant-associated Pseudomonas spp.: diversity, activ-ity, biosynthesis, and regulation. Mol Plant Microbe Interact 19(7):699–710. doi:10.1094/MPMI-19-0699

Raaijmakers JM, De Bruijn I, Nybroe O, Ongena M (2010) Naturalfunctions of lipopeptides from Bacillus and Pseudomonas: morethan surfactants and antibiotics. FEMS Microbiol Rev 34(6):1037–1062. doi:10.1111/j.1574-6976.2010.00221.x

Rogers J, Dowsett AB, Dennis PJ, Lee JV, Keevil CW (1994) Influenceof temperature and plumbing material selection on biofilm forma-tion and growth of Legionella pneumophila in a model potable watersystem containing complexmicrobial flora. Appl EnvironMicrobiol60(5):1585–1592

Sabate DC, Audisio MC (2013) Inhibitory activity of surfactin, producedby different Bacillus subtilis subsp. subtilis strains, against Listeriamonocytogenes sensitive and bacteriocin-resistant strains. MicrobiolRes 168(3):125–129. doi:10.1016/j.micres.2012.11.004

Sabate DC, Carrillo L, Audisio MC (2009) Inhibition of Paenibacilluslarvae and Ascosphaera apis by Bacillus subtilis isolated fromhoneybee gut and honey samples. Res Microbiol 160(3):193–199.doi:10.1016/j.resmic.2009.03.002

Schuster FL (2002) Cultivation of pathogenic and opportunistic free-living amebas. Clin Microbiol Rev 15(3):342–354

Sen R (2010) Surfactin: biosynthesis, genetics and potential applications.Adv Exp Med Biol 672:316–323

Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses andspecific functions. Mol Microbiol 56(4):845–857. doi:10.1111/j.1365-2958.2005.04587.x

Steinert M, Hentschel U, Hacker J (2002) Legionella pneumophila: anaquatic microbe goes astray. FEMS Microbiol Rev 26(2):149–162

Symmank H, Franke P, Saenger W, Bernhard F (2002) Modification ofbiologically active peptides: production of a novel lipohexapeptideafter engineering of Bacillus subtilis surfactin synthetase. ProteinEng 15(11):913–921

Tang JS, Zhao F, Gao H, Dai Y, Yao ZH, Hong K, Li J, Ye WC, Yao XS(2010) Characterization and online detection of surfactin isomersbased on HPLC-MS(n) analyses and their inhibitory effects on theoverproduction of nitric oxide and the release of TNF-alpha and IL-6 in LPS-induced macrophages. Mar Drugs 8(10):2605–2618. doi:10.3390/md8102605

Temmerman R, Vervaeren H, Noseda B, Boon N, Verstraete W (2007)Inhibition of Legionella pneumophila byBacillus sp. Eng Life Sci 7:497–503

Thomas V, Bouchez T, Nicolas V, Robert S, Loret JF, Levi Y (2004)Amoebae in domestic water systems: resistance to disinfectiontreatments and implication in Legionella persistence. J ApplMicrobiol 97(5):950–963. doi:10.1111/j.1365-2672.2004.02391.x

Uppalapati SR, Ishiga Y, Wangdi T, Kunkel BN, Anand A, Mysore KS,Bender CL (2007) The phytotoxin coronatine contributes to patho-gen fitness and is required for suppression of salicylic acid accumu-lation in tomato inoculated with Pseudomonas syringae pv. tomatoDC3000. Mol Plant Microbe Interact 20(8):955–965. doi:10.1094/MPMI-20-8-0955

Verdon J, Berjeaud JM, Lacombe C, Hechard Y (2008) Characterizationof anti-Legionella activity of warnericin RK and delta-lysin I fromStaphylococcus warneri. Peptides 29(6):978–984. doi:10.1016/j.peptides.2008.01.017

Vollenbroich D, Pauli G, Ozel M, Vater J (1997) Antimycoplasma prop-erties and application in cell culture of surfactin, a lipopeptideantibiotic from Bacillus subtilis. Appl Environ Microbiol 63(1):44–49

Wingender J, Flemming HC (2011) Biofilms in drinking water and theirrole as reservoir for pathogens. Int J Hyg Environ Health 214(6):417–423. doi:10.1016/j.ijheh.2011.05.009

Xu HM, Rong YJ, Zhao MX, Song B, Chi ZM (2014) Antibacterialactivity of the lipopetides produced by Bacillus amyloliquefaciensM1 against multidrug-resistant Vibrio spp. isolated from diseasedmarine animals. Appl Microbiol Biotechnol 98(1):127–136. doi:10.1007/s00253-013-5291-1

Zhao Y, Thilmony R, Bender CL, Schaller A, He SY, Howe GA (2003)Virulence systems of Pseudomonas syringae pv. tomato promotebacterial speck disease in tomato by targeting the jasmonate signal-ing pathway. Plant J 36(4):485–499

Appl Microbiol Biotechnol

Author's personal copy