Antimicrobial effect of diallyl sulphide on Campylobacter jejuni biofilms

Antimicrobial effect of diallyl sulphide onCampylobacter jejuni biofilms

Xiaonan Lu1–3, Derrick R. Samuelson1, Barbara A. Rasco2 and Michael E. Konkel1*

1School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7520, USA;2School of Food Science, Washington State University, Pullman, WA 99164-6376, USA; 3Key Laboratory of Food Nutrition and Safety,

Ministry of Education of China, Tianjin University of Science and Technology, Tianjin 300457, China

*Corresponding author. Tel: +1-509-335-5039; Fax: +1-509-335-4159; E-mail: [email protected]

Received 27 January 2012; returned 28 February 2012; revised 9 March 2012; accepted 20 March 2012

Objectives: Bacterial biofilms pose significant food safety risks because of their attachment to fomites and foodsurfaces, including fresh produce surfaces. The purpose of this study was to systematically investigate theactivity of selected antimicrobials on Campylobacter jejuni biofilms.

Methods: C. jejuni biofilms and planktonic cells were treated with ciprofloxacin, erythromycin and diallylsulphide and examined using infrared and Raman spectroscopies coupled with imaging analysis.

Results: Diallyl sulphide eliminated planktonic cells and sessile cells in biofilms at a concentration that was atleast 100-fold less than used for either ciprofloxacin or erythromycin on the basis of molarity. Distinct cell lysiswas observed in diallyl sulphide-treated planktonic cells using immunoblot analysis and was confirmed by arapid decrease in cellular ATP. Two phases of C. jejuni biofilm recalcitrance modes against ciprofloxacin anderythromycin were validated using vibrational spectroscopies: (i) an initial hindered adsorption into biofilmextracellular polymeric substance (EPS) and delivery of antibiotics to sessile cells within biofilms; and (ii) adifferent interaction between sessile cells in a biofilm compared with their planktonic counterparts. Diallylsulphide destroyed the EPS structure of the C. jejuni biofilm, after which the sessile cells were killed in asimilar manner as planktonic cells. Spectroscopic models can predict the survival of sessile cells within biofilms.

Conclusions: Diallyl sulphide elicits strong antimicrobial activity against planktonic and sessile C. jejuni and mayhave applications for reducing the prevalence of this microbe in foods, biofilm reduction and, potentially, as analternative chemotherapeutic agent for multidrug-resistant bacterial strains.

Keywords: antimicrobial mode, antibiotic, bioanalytical spectroscopy, Raman imaging

IntroductionCampylobacter jejuni is a leading cause of bacterial gastrointes-tinal disease worldwide. C. jejuni-mediated disease (campylobac-teriosis) is normally self-limiting, but in some instances isassociated with severe enteritis, septicaemia and a higherincidence of Guillain–Barre syndrome.1 In severe cases ofcampylobacteriosis, erythromycin (a macrolide) or ciprofloxacin(a fluoroquinolone) are commonly used for treatment.2

However, fluoroquinolone resistance is common amongst C. jejunistrains and macrolide resistance in strains recovered from clinicalisolates has appeared in recent years.2,3 Only through the devel-opment of new antimicrobial agents will it be possible to treatthe growing number of multidrug-resistant bacteria.4

C. jejuni may form a monospecies biofilm.5 – 7 Adherent cells areembedded within an extracellular polymeric substance (EPS)

composed of polysaccharides, proteins, nucleic acids, lipids andhumic-like substances. The EPS matrix mediates cell/cell commu-nications such as quorum sensing, maintains biofilm hydrationand protects microorganisms against environmental stresses,including antibiotic treatments.8 The chemical composition andstructure of EPS varies markedly, depending upon severalfactors: cell species, metabolic activity, nutrient availability,biofilm maturity level and physicochemical conditions.9

In situ studies indicate that bacteria in biofilms are as muchas 1000 times more resistant to antibiotics than planktoniccells.10 – 12 The factors contributing to antibiotic resistanceinclude altered physiological growth state and altered microen-vironments that impede penetration of the antibiotic into thebiofilm.13,14

New spectroscopic techniques have made it possible tostudy the properties of microbial biofilms without the

# The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.For Permissions, please e-mail: [email protected]

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confounding effects introduced by staining techniques. Forexample, traditional fluorescence-induced confocal laser scan-ning microscopy (CLSM) requires staining. Coupling eitherFourier transform infrared (FT-IR) spectroscopy or Raman spec-troscopy with confocal microscopy can provide detailed chem-ical information about microbial cells and complex biofilmmatrices without staining. These two spectroscopic methodsprovide complementary analytical techniques using ‘whole-organism fingerprint’ spectral features of bacteria with spatialresolution in the micrometre range, enabling correlationsbetween optical and chemical images.15 – 17 The biochemicalproperties of healthy and injured microbial cells have beenstudied by infrared and Raman spectroscopy,18 – 25 but therehave been few studies investigating biofilm properties.26 – 31 Re-cently, a number of studies have been conducted using vibra-tional spectroscopy to monitor the inactivation of planktonicbacteria exposed to antibiotics.4,32 – 38

Recent studies indicate that plants, specifically Allium spp.,contain antimicrobial agents such as diallyl sulphide that arehighly effective against major foodborne pathogens.39,40 Wehypothesized that diallyl sulphide might be more effective in in-activating bacterial biofilms than erythromycin or ciprofloxacinbased on its ability to freely penetrate the phospholipid bilayersof bacterial cell walls.39,40 The objectives of this study were tocompare systematically the effectiveness of diallyl sulphidewith antibiotics commonly used to treat campylobacteriosis.The novelty of this study is that researchers have not examinedthe antimicrobial activity of diallyl sulphide against any type ofbacterial biofilms, including C. jejuni. Moreover, we used biophys-ical and biochemical techniques to investigate the differences inantimicrobial mechanisms of diallyl sulphide and antibioticsagainst C. jejuni biofilms. We show for the first time that the anti-microbial activity of diallyl sulphide against C. jejuni planktoniccells and biofilms is much greater than that of selective antibio-tics. In addition, we are the first to use vibrational spectroscopyto validate that C. jejuni planktonic cells have a different inter-action mode with antibiotics compared with their sessile cellcounterparts after biofilm EPS has been destroyed. Planktoniccells and sessile cells have a similar mode of susceptibility todiallyl sulphide, and it was much easier to destroy biofilm EPSwith diallyl sulphide than with antibiotics.

Materials and methods

Chemicals and reagentsCiprofloxacin (purity 98%), erythromycin (purity 95%) and diallylsulphide (purity 98%) were purchased from Sigma. The purity andstability of diallyl sulphide were determined using methods described ina previous study.39

Bacterial strains and culture methodsCampylobacter jejuni strains F38011 and NCTC 11168 were used in thisstudy. Bacterial cells were cultured either on Mueller–Hinton agarplates supplemented with 5% citrated bovine blood (MHB) or inMueller–Hinton broth with constant shaking. C. jejuni were incubated ina microaerobic environment (85% N2, 10% CO2, 5% O2) at 378C, andwere passaged every 48 h.

Biofilm formationOvernight cultures of C. jejuni isolates were diluted to an OD540 of 0.03(�107 cfu/mL) and 100 mL was added to the surface of a sterile nitrocel-lulose membrane (0.45 mm pore size, 47 mm diameter; SartoriusStedim-type filters). The membranes were placed onto agar plates andincubated in a microaerobic environment at 378C. The membraneswere aseptically transferred to new agar plates every 24 h for up to3 days to form discernible and uniform C. jejuni biofilms with a surfacearea of �1 cm2.

Antimicrobial treatments with diallyl sulphideand selected antibioticsSelected antibiotics [100 mg/L (equivalent to 300 mM) ciprofloxacin and100 mg/L (equivalent to 136 mM) erythromycin] and/or diallyl sulphide(0.1 mg/L, equivalent to 1 mM) were added to 5 mL bacterial culturegrown overnight (�109 cfu/mL) and incubated for different times (0, 1,3, 5, 8, 10, 12 and 24 h). The concentration of antibiotics used wasbased on previous studies.41 At each sampling time, the bacterial sus-pensions were serially diluted and spread onto the surface of MHB forenumeration of viable bacteria. For the biofilm experiments, eachbiofilm was treated with the same antimicrobial agents as mentionedabove. Bacterial biofilms coated on nitrocellulose membrane were asep-tically removed from MHB agar plates, then placed into 20 mL of MHbroth with the same selective concentration of antimicrobial agent(100 mg/L ciprofloxacin, 100 mg/L erythromycin or 0.1 mg/L diallyl sul-phide) and incubated with gentle shaking in a microaerobic environmentat 378C. Untreated bacterial biofilms maintained intact EPS and viablesessile cell numbers within biofilms during culture for 24 h. At each sam-pling time (0, 1, 3, 5, 7, 12 and 24 h), the recovered biofilms were rinsedtwice with PBS to remove any residual antimicrobial agents.

Bacterial biofilms were detached using a solution of 0.1% trypsin(25 mL) for 15 min. This treatment did not affect cell viability (data notshown). Following incubation, the trypsin solution was collected, seriallydiluted and spread onto the surface of MHB for enumeration of viablebacteria.

The MICs of ciprofloxacin, erythromycin and diallyl sulphide againstC. jejuni planktonic cells were determined using a broth microdilutionmethod. Briefly, serial 2-fold dilutions of antimicrobials were preparedin a 96-well microtitre plate using MH broth. Freshly grown bacterialcells at log phase were inoculated into each well to give a finalconcentration of �105 cfu/mL. After the inocula had been incubatedin a microaerobic environment for 24 h at 378C, the growth of the bac-teria in each well was determined by measuring the absorbance atOD540 and compared with the growth of the positive control (a wellcontaining no antimicrobials). The MIC was recorded as the lowestconcentration of antimicrobial that completely inhibited bacterialcell growth.

Determination of cytosolic ATPThe ATP Bioluminescence Assay kit CLS II (Roche) was used according tothe manufacturer’s recommendations to analyse the influence of diallylsulphide, ciprofloxacin and erythromycin on C. jejuni intracellular ATPlevels. Bacterial samples (2 mL, �109 cfu/mL) were diluted with deio-nized water to an appropriate ATP concentration (1025 to 10210 MATP). Then, 50 mL of each sample was transferred to a microtitre platethat was kept on ice until measurement. Luciferase reagent (50 mL)was added and luminescence read with a VICTORTM X5 Multilabel PlateReader (PerkinElmer) at 208C.

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SDS–PAGE and immunoblot analysisC. jejuni F38011 and NCTC 11168 wild-type strains (�109 cfu/mL) weretreated with 0.1 mg/L diallyl sulphide. Supernatants from these culturesand from untreated controls were obtained following low-speed centrifu-gation (6000 g) and analysed by SDS–PAGE using standard procedures.For immunoblot analysis, the membranes were incubated with a rabbitanti-C. jejuni polyclonal serum prepared in PBS/Tween [20 mM sodiumphosphate and 150 mM sodium chloride, pH 7.5, containing 0.01%(v/v) Tween 20] with 9% non-fat dry milk. The membranes were thenincubated with horseradish peroxidase-conjugated goat anti-rabbitimmunoglobulin G (whole molecule) diluted 1:1000 in PBS/Tween anddeveloped using Western Lightning chemiluminescence (PerkinElmer)according to the manufacturer’s directions.

Scanning electron microscopy (SEM)SEM was employed to examine changes in C. jejuni biofilms untreatedand treated with 0.1 mg/L diallyl sulphide for 1 h in a microaerobic envir-onment at 378C. The C. jejuni biofilm was fixed with 2% glutaraldehyde,2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) overnight at48C. The fixed biofilms were then washed with phosphate buffer, post-fixed in 1% osmium tetroxide for 2 h at 208C and rinsed twice with0.1 M phosphate buffer prior to dehydration in an ethanol series. Theywere then incubated with 100% hexamethyldisilazane (HMDS) overnightand air dried. These prepared biofilms were mounted onto SEM stubs andsputter coated with a thin layer of gold. The coated samples wereobserved under an FEI Quanta 200F scanning electron microscopeusing an accelerating voltage of 30 kV.

FT-IR spectroscopy analysisAt the end of each treatment, 2 mL of each planktonic bacterial culture(�109 cfu/mL) was recovered by centrifugation at 8000 g for 10 min at208C. The supernatant was discarded and the pellet resuspended in20 mL PBS to remove components of the medium. Then, 20 mL was fil-tered through an aluminium oxide membrane filter (0.2 mm pore size,25 mm OD) (Anodisc) using a Whatman vacuum glass membrane filterholder to recover bacterial cells. The Anodisc filters were air-driedunder laminar flow at 208C for 60 min. For the biofilm experiment, thenitrocellulose membrane with a biofilm was removed from the agarplate on which it was cultured and placed directly on the diamondcrystal cell of a Nicolet 380 FT-IR spectrophotometer (Thermo Electron)for spectral measurement. In agreement with previous work, the nitro-cellulose membrane did not interfere with the collection of high-qualityspectra.42 FT-IR spectroscopy was used to collect sample spectral fea-tures for each sample in triplicate, as described in previous studies.39,40

Raman spectroscopic analysisKlariteTM (Renishaw Diagnostics) surface-enhanced Raman scattering(SERS)-active substrates were used for planktonic bacterial samples toenhance the intensities of Raman signals. Untreated and treated plank-tonic C. jejuni cells (10 mL) were deposited onto the substrate andRaman measurements were taken after drying for 1 h under a fumehood at 208C. For the biofilm experiments, the nitrocellulose membranescoated with the bacterial biofilms were placed directly under a micro-scope for focus adjustment and employed for Raman spectral collection.

Raman spectroscopic analysis was performed using a WITecalpha300 Raman microscope (WITec, Ulm, Germany) equipped with aUHTS-300 spectrometer and a 532 nm laser delivering �2 mW incidentlaser power on the planktonic bacterial samples and �0.2 mW incidentlaser power on the biofilm samples through a 100× objective(Nikon). Raman scattering spectra were collected by a 1600×200 pixel

charge-coupled device (CCD) array detector. The size of each pixel was16 ×16 mm. WITec Control v1.5 software (WITec, Ulm, Germany) wasemployed for instrumental control and data collection. Raman spectraof each planktonic bacterial and/or biofilm sample were collected overa simultaneous wavenumber shift range of 3700–200 cm21 in extendedmode. For measurement at a single location, each full spectral measure-ment was conducted with a 3 s integration time with 20 spectralaccumulations (total integration time 60 s). Eight spectra were collectedfor each sample in triplicate.

In situ Raman mapping analysisFull area raster scans were performed to create Raman maps, withsingle-spectrum integration times of 3 s, saving 600 spectra acquiredover a regularly spaced array of sample locations in a grid pattern(30 ×20 arrays). For image resolution the numerical aperture was 0.95,which gave a spatial resolution of �345 nm. A computer-controlled xyzmotorized stage was employed during Raman map generation. Subse-quent band analysis for the collected spectra was applied to createintensity-correlated maps for relevant Raman bands. Thus, chemicalimages corresponding to optical images of the biofilm were generatedbased upon the area beneath the baseline-corrected specific bands. Anestimate of the distribution and concentration of biochemical constitu-ents within a section of the biofilm could be determined subsequently(N¼15).

Spectroscopic-based chemometric analysisASCII data pertaining to individual Raman spectra were exported fromWITec Project software. Infrared and Raman spectra were initially prepro-cessed using OMNIC (Thermo Electron). The spectra were subtractedfrom relative background if necessary. Automatic baseline correctionwas then employed to flatten the baseline, followed by smoothingusing a Gaussian function with a bandwidth of 9.643 cm21. The heightand area of vibrational spectral bands were measured and calculatedusing OMNIC and Matlab. Second-derivative transforms of vibrationalspectra using a nine-point Savitzky–Golay filter and wavelet transformswith a scale of 7 were performed in Matlab. The reproducibility of infraredand Raman spectra was determined by calculating Dy1y2 as described inprevious studies.39,40 Partial least squares regression (PLSR) models wereestablished based upon processed vibrational spectra and employed forquantitative analyses in Matlab. A total of four spectra of each samplewere used to establish the calibration model (N¼30). A ‘leave-one-out’cross-validation was employed to study the predictive power of thePLSR chemometric model by removing vibrational spectra for onesample from the dataset and applying a calibration to the remainingsamples.40 The overall suitability of the developed chemometricmodels for predicting live C. jejuni numbers in the biofilm was evaluatedusing residual prediction deviation (RPD) values and other parametersdescribed in previous studies.25,39,40

Statistical analysisEach experiment was independently repeated a minimum of three timesto ensure reproducibility. The results were expressed as the mean of threeindependent replicates + standard deviation. The significance of differ-ences between band heights of raw and second derivative transformedspectra was determined using one-way analysis of variance followingt-test in Matlab to determine critical variations of chemical componentsof samples. Differences were considered significant at P,0.05.

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Results and discussion

Inhibitory effects of diallyl sulphide and antibioticson C. jejuni planktonic and sessile cells in biofilm

In the current study, we used C. jejuni planktonic cells as the controlto determine and compare the effectiveness of diallyl sulphide andtwo selective antibiotics, ciprofloxacin and erythromycin, in inacti-vating C. jejuni biofilms. In addition, we hypothesized that C. jejuniplanktonic cells and sessile cells in biofilms have different interactionmodes and susceptibility mechanisms to treatment with diallyl sul-phide and antibiotics. We used planktonic cells as a control through-out our experiments to investigate the antimicrobial mechanism ofthese antimicrobials. Two strains of C. jejuni were used, strainF38011 (a human clinical isolate) and NCTC 11168 (a sequencedstrain). The MIC values of ciprofloxacin for these strains were identi-cal (16 mg/L), but the strains differed in their susceptibility to eryth-romycin. The MIC for C. jejuni F38011 was 8 mg/L and that for strainNCTC 11168 was 4 mg/L. An MIC of 0.04 mg/L was established fordiallyl sulphide. The survival of planktonic and sessile cells treatedwith these antimicrobial agents, at concentrations exceedingthese MICs, was examined over a 24 h time period (Figure 1).Diallyl sulphide eliminated planktonic cells and sessile cellsof both strains much faster than the antibiotics did, at aconcentration that was 136- to 300-fold less than used for erythro-mycin or ciprofloxacin, respectively.

The survival of the C. jejuni F38011 and NCTC 11168 biofilmstreated with antimicrobial agents for 24 h are also shown in

Figure 1. Sessile cells exhibited greater resistance to treatmentwith ciprofloxacin and erythromycin than planktonic cells, dem-onstrating the recalcitrant properties of the biofilm (Figure 1).Diallyl sulphide treatment totally inactivated the cells withinthe biofilm within 5 h compared with .24 h required for cipro-floxacin and erythromycin, as determined by the number ofviable bacteria recovered following treatment. This is the firsttime diallyl sulphide has been shown to have a significantlyhigher antimicrobial effect against bacterial biofilms comparedwith commonly used antibiotics. Furthermore, these data alsosuggest that the interaction mode of antimicrobial action ofdiallyl sulphide is different from that of the two antibiotics.

Inhibition of cellular metabolism—cytosolic ATP levels

To address the antimicrobial mechanism of diallyl sulphideagainst C. jejuni, we first studied its antimicrobial mechanismagainst planktonic bacterial cells. Cellular ATP levels correlatewith cell viability. After a loss of membrane integrity, cells losethe capacity to synthesize ATP, and endogenous ATPasesdestroy any remaining ATP; thus, ATP levels drop precipitously.ATP levels of C. jejuni treated with 0.1 mg/L diallyl sulphidedecreased markedly after 1.5 h of treatment (Figure S1, availableas Supplementary data at JAC Online). However, the ATP levels ofC. jejuni treated with either 100 mg/L ciprofloxacin or 100 mg/Lerythromycin remained stable following a 12 h treatment(Figure S2, available as Supplementary data at JAC Online).

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Figure 1. Viable C. jejuni planktonic cells and sessile cells in biofilms are eradicated by antimicrobials. Survival curves of untreated bacteria (black lines)and bacteria treated with 100 mg/L ciprofloxacin (black dotted lines), 100 mg/L erythromycin (grey lines) and 0.1 mg/L diallyl sulphide (grey dottedlines) in a microaerobic environment at 378C. Panels (a) and (b) show data for planktonic cells and panels (c) and (d) show data for sessile cells inbiofilm. Strain C. jejuni NCTC 11168 is shown in panels (a) and (c) and strain F38011 is shown in panels (b) and (d).

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The initial increase in ATP levels observed in C. jejuni treated withciprofloxacin is possibly due to the inhibition of cell division andthe resulting increase in the volume of individual cells. In add-ition, neither ciprofloxacin nor erythromycin is known to com-promise bacterial membrane integrity. Thus, a drop in ATP isnot expected for these compounds until the cells are no longerviable. Taken together, these results demonstrate that diallylsulphide decreases cytosolic ATP and subsequently inhibitscellular metabolism and inactivates bacterial cells comparedwith ciprofloxacin and erythromycin.

Planktonic cell lysis by diallyl sulphide is validatedby immunoblot analysis

There are several possible mechanisms to explain the decline inbacterial viability observed upon treatment with diallyl sulphide,

including bacterial cell lysis, inactivation of key metabolic pro-teins and inhibition of protein synthesis. Treatment of planktoniccells with 0.1 mg/L diallyl sulphide resulted in cell lysis or leakage,as judged by immunoblot analysis (Figure S3, available asSupplementary data at JAC Online).

Infrared and Raman spectral features of C. jejuni biofilm

Infrared and Raman spectral features of C. jejuni planktoniccells have been reported previously.18,39 Figure 2 shows anoptical microscope image and typical Raman spectra of asingle-species biofilm (C. jejuni NCTC 11168) obtained at a532 nm excitation wavenumber and a 60 s integration time.Spectral features associated with polysaccharides, nucleicacids and proteins are apparent in the Raman spectra forthese biofilms and are impacted by antimicrobial treatment.

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Figure 2. Characterization of C. jejuni biofilm EPS reveals chemical composition. (a) Optical image of C. jejuni NCTC 11168 biofilm with the mappingarea for Raman spectroscopy marked with a frame. (b) Raman maps for the band at 868 cm21, corresponding to monosaccharides andpolysaccharides. (c) Spectra for maps were collected for a period of 60 s from a 10 ×10 mm biofilm area (black line, position 1; blue line, position2; red line, position 3; green line, position 4).


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The methods employed here provided greater detail of the bio-chemical features of bacterial biofilms than recent studies inwhich multispecies biofilms had been characterized usingsurface-enhanced Raman spectroscopy.26,27 Raman and SERSspectral features reflect similar chemical properties, but donot necessarily coincide due to chemical enhancemmenteffects that may cause band shifts. Interaction between specif-ic analytes in biofilm and SERS active surface (hot spots) mayalso cause variations from band shifts.20,22,27 The FT-IR and

Raman band assignments for C. jejuni biofilm are summarizedin Table S1 (available as Supplementary data at JAC Online)and a comparison of FT-IR spectral features for the C. jejunibiofilm and planktonic cell is shown in Figure S4 (available asSupplementary data at JAC Online). The chemical compositionof biofilm could be determined using FT-IR and Raman spectro-scopies. In addition, we demonstrated that the protein andpolysaccharide compositions of C. jejuni planktonic cells andbiofilms were different in the present study.

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Figure 3. Interaction mode between C. jejuni planktonic cells with antimicrobial agents studied by FT-IR and Raman spectroscopy. (a)Second-derivative transformation of FT-IR spectral features to illustrate variations of untreated C. jejuni planktonic cells (black line) and cellstreated with 100 mg/L ciprofloxacin (red line), 100 mg/L erythromycin (green line) and 0.1 mg/L diallyl sulphide (blue line) for 8 h in a microaerobicenvironment at 378C. (b) Second-derivative transformation of Raman spectral features to illustrate variations of untreated C. jejuni planktonic cells(black line) and cells treated with 100 mg/L ciprofloxacin (red line), 100 mg/L erythromycin (green line) and 0.1 mg/L diallyl sulphide (blue line) for8 h in a microaerobic environment at 378C.

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Reproducibility of Raman mapping and vibrationalspectroscopy for C. jejuni biofilm

Spectral reproducibility is critical and is the precondition forchemometric statistical analyses, such as second-derivative trans-formation and its comparison. The distribution of chemical com-ponents on biofilms will also affect spectral reproducibility andthis distribution can be determined using spectroscopy-basedmapping techniques. Thus, we determined the different chemicalcomponents of the C. jejuni biofilm matrices using confocalRaman mapping (Figure 2). The spectra from a specific biofilmarea were obtained by raster scans. Raster scanning is a techniquefor capturing a video rectangular pattern of an image line by line.Raman maps of the relevant band intensities were calculated andcorrelated with the regions on the optical images of the biofilmfrom which they were taken. Figure 2(b) shows the Raman mapof the band intensity at 868 cm21 collected from an area of10×10 mm (see black frame in Figure 2a). This Raman mapshows the distribution of monosaccharide/polysaccharide in theupper biofilm layer. Previous work has demonstrated that the dis-tribution of proteins and polysaccharides in the biofilm can bemapped at other wavelength regions with marker bands selectedfor the relevant biological substances.27 However, being able tocompare experimental results is dependent upon being able toobtain a reliable estimate of reproducibility.

The reproducibility of vibrational spectra from independentexperiments and various sample locations was calculated asDy1y2 values. Mean D values between 7 and 10 are considered

to be normal when analyzing the first- or second-derivativetransforms of spectral features from samples prepared in inde-pendent assays.18,39,40 Others have asserted that D values canbe as high as 300 when microorganisms from different generaare compared,18 but there are no reported studies by other inves-tigators calculating spectral reproducibility for bacterial biofilms.Wavenumber and cultivation time were critical to the reproduci-bility of biofilm vibrational spectra, the same as for bacterialplanktonic cells.18,39,40 The wavenumber region for Ramanspectra of C. jejuni biofilms was from 1800 to 400 cm21 whilethe wavenumber region for FT-IR spectra of C. jejuni biofilmsranged from 1800 to 700 cm21 (‘fingerprint’ region). TheD value for Raman spectra was 7.19+1.12 to 9.10+1.56 andthe D value for FT-IR spectra was 16.96+2.25 to 21.39+3.92after 72 h of culture on MHB agar. Both FT-IR and Ramanspectra offer high reproducibility, with the reproducibility ofRaman spectra being significantly (P,0.05) greater than thatfor FT-IR spectra. While physiological heterogeneity is commonin biofilms,43 we found that the heterogeneity did not significant-ly affect (P.0.05) vibrational spectral reproducibility. Collectively,our data show high spectral reproducibility and this is critical forthe development of reliable chemometric models.44

Mode of C. jejuni planktonic cell inactivationby antimicrobial agents

For the second-derivative transformation analyses of FT-IR andRaman spectra, the planktonic cells were treated with ciprofloxacin,

Table 1. Assignment of significant (P,0.05) FT-IR and Raman band variations of C. jejuni planktonic cells treated with 100 mg/L ciprofloxacin,100 mg/L ciprofloxacin or 0.1 mg/L diallyl sulphide for 8 h in a microaerobic environment at 378C

Treatment/FT-IRfrequency (cm21) Assignment

Raman frequency(cm21) Assignment

Ciprofloxacin 100 mg/L800 left-handed helix DNA 788 C5-O-P-O-C3 phosphodiester bands in DNA1084 phosphate/sugar backbone of nucleic acids 828 O-P-O stretch DNA1220 stretching phosphate antisymmetric

vibration in B-form DNA1093 symmetric PO2 stretching vibration of the DNA

backbone1606 adenine vibration in DNA 1243 phosphodiester groups of nucleic acids

1458 nucleic acid mode

Erythromycin 100 mg/L1400 symmetric CH3 bending modes of methyl

groups of proteins760 ring breathing tryptophan

1455 symmetric bending modes of methylgroups in skeletal proteins

1004 phenylalanine

1655 amide I (a-helix) 1260 amide III vibration mode of structural proteins

Diallyl sulphide 0.1 mg/L916 phosphodiester 524 S-S disulphide stretching in proteins991 phosphodiester 540 y (S-S) trans-gauche-trans (amino acid cysteine)1220 stretching phosphate antisymmetric

vibration in B-form DNA760 ring breathing tryptophan

1515 amide II 1131 fatty acid1545 amide II 1323 guanine1637 amide I 1470 C¼N stretching1655 amide I


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erythromycin and diallyl sulphide for 8 h and the bacterial spectralvariations compared with one another (Figure 3a and b). For C.jejuni planktonic cells treated with 100 mg/L ciprofloxacin, signifi-cant (P,0.05) band variations were related to DNA/RNA informa-tion. Table 1 provides the detailed identification for FT-IR andRaman band variations. Consistent with previous work, we demon-strated from spectral measurement that ciprofloxacin alters DNAstructure in C. jejuni planktonic cells.

For C. jejuni planktonic cells treated with 100 mg/L erythromy-cin, significant (P,0.05) band variations were related to thesecondary structure of proteins. Table 1 provides identificationfor FT-IR and Raman bands. Spectral variations in secondarystructure of protein regions found in this study confirm thatbacterial protein synthesis and inhibition of processes criticalfor cell function or replication are inhibited by macrolides thatbind to the 50S subunit of the bacterial 70S rRNA complex.2

For C. jejuni planktonic cells treated with 0.1 mg/L (1 mM)diallyl sulphide, significant (P,0.05) band variations wereobserved in the whole fingerprint wavenumber regions for boththe FT-IR and Raman spectra. Table 1 provides the identificationfor FT-IR and Raman band variations. The spectral variations inthe sulphur region determined by Raman spectroscopy are inagreement with previous studies using higher concentrations ofdiallyl sulphide (5, 10 and 20 mM) for different treatmenttimes,39,40 and suggest that inhibition of certain thiol-containingenzymes/proteins in C. jejuni by the rapid reaction of diallyl sul-phide with thiol groups may be an important mechanism forantimcrobial activity. The transmembrane transfer of sulphur-containing compounds into C. jejuni could be monitored usingRaman spectroscopy, but not FT-IR spectroscopy, emphasizingthe importance of using complementary vibrational spectrosco-pies at the same time for studies of bacterial resistance andinactivation against antimicrobial agents.

C. jejuni biofilm EPS recalcitrance mode againstantimicrobial agents

Biofilms show greater recalcitrance towards antimicrobial agentswhen compared with planktonic cells.34 Recalcitrance is reflectedin the infrared spectral features of C. jejuni biofilm that weredifferent from those of planktonic cells at three wavenumbers(Figure S4). Specifically, wavenumber differences wereobserved in the amide III band components of proteins (band1280 cm21), the ring structure of polysaccharides (band1162 cm21) and the C2 endo conformation of polysaccharides(band 829 cm21).16 These three bands reflect the properties ofthe major components of biofilm EPS5,8 and were selected tomonitor biofilm EPS variations in response to antibiotic anddiallyl sulphide treatment.

We observed no structural difference in the C. jejuni biofilmEPS after ciprofloxacin (100 mg/L) treatment for approximately7 h and erythromycin (100 mg/L) treatment for �5 h. However,the EPS was totally destroyed by diallyl sulphide (0.1 mg/L) treat-ment within 1 h (Figure 4). FT-IR spectral variations at 1280,1162 and 829 cm21 indicated recalcitrance of the biofilm EPSstructure to ciprofloxacin and erythromycin, showing that EPSprovides a physical or chemical diffusion barrier and altersadsorption properties to antimicrobial penetration into thebiofilm, preventing the access of antibiotics to the embeddedsessile cells.11 – 14 Reaction of antibiotics with or adsorption to

biofilm components can limit transport into the cells in abiofilm.14,45 Suci et al.32 used FT-IR spectroscopy to monitorthe impeded transport of ciprofloxacin to the biofilm–substratum interface and direct interaction of biofilm compo-nents with the antibiotic in a flowing system. Similarly, preven-tion of diffusion of piperacillin into Pseudomonas aeruginosabiofilms has been observed.46 In addition, increasing the size ofthe hydrophobic side chains of selected quaternary ammoniumcompounds reduced the susceptibility of Staphylococcus aureustreated with antibiotics when these bacteria were embedded ina hydrophobic EPS matrix.47 Biofilm EPS is not impenetrable toantibiotics, but transport is impeded.12 We demonstrated thatthe biofilm EPS was intact during antibiotic treatment duringthe first several hours and that there was a transport delay ofantibiotic compounds to the sessile bacterial cells within the

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Figure 4. Treatment of C. jejuni biofilm with diallyl sulphide causes greateralterations in EPS proteins and polysaccharides than ciprofloxacin anderythromycin as evidenced by FT-IR spectroscopic analysis. Shown arethe variations of specific FT-IR bands related to biofilm untreated EPS(black lines) and EPS treated with 100 mg/L ciprofloxacin (black dottedlines), 100 mg/L erythromycin (grey lines) and 0.1 mg/L diallyl sulphide(grey dotted lines). (a) 1280 cm21, amide III band components ofproteins. (b) 1162 cm21, ring structure of polysaccharides. (c) 829 cm21,C2 endo conformation of polysaccharide.

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biofilm. The biofilm EPS began to decay after this initial recalci-trance (Figure 4). However, given the biofilm survival curvesshown in Figure 1, other mechanisms must also be acting tosupport biofilm cell survival besides diffusion limitations.

The biofilm structure was destroyed by 0.1 mg/L diallyl sul-phide within 1 h, explaining why diallyl sulphide had a morepowerful antimicrobial effect on C. jejuni biofilm compared withthe antibiotics tested at the same concentrations. It is knownthat organosulphur compounds can freely penetrate throughthe phospholipid bilayers of bacterial cell walls48 and causeplanktonic cell lysis (Figure S3). We hypothesize that organosul-phur compounds destroy the biofilm EPS structure more easilythan erythromycin and ciprofloxacin because diallyl sulphide ismore polar, smaller and hydrophilic. This biological profile pro-vides a faster penetration of diallyl sulphide through biofilmEPS to sessile cells. Furthermore, disaggregation of a biofilmwill increase the effectiveness of an antimicrobial agent.11

What remains to be determined is whether diallyl sulphidecauses the EPS to detach from the biofilm structure or whetherthe EPS is eliminated by other means.

C. jejuni sessile cells in biofilm had a differentinteraction mode with antimicrobial agents comparedwith planktonic cells

C. jejuni biofilm EPS was totally destroyed after 12 h of treatmentwith antimicrobial agents (Figure 4) and subsequently providedsessile cells with little protection. Significant (P,0.05) bandvariations in Raman spectra between treated and untreatedsessile cells were determined (Figure 5). Ciprofloxacin- anderythromycin-treated sessile cells show band variations at the

same three wavenumbers: ring breathing tryptophans(760 cm21),16 CH3CH2 wagging mode in purine bases ofDNA (1324 cm21)17 and bending modes of methyl groups(1401 cm21).17 Compared with Figure 3(b), sessile cells in abiofilm had a different interaction mode with antibiotics com-pared with planktonic cells, as reflected in band variations atdifferent wavenumbers. In addition, sessile cells in the biofilmwere more resistant to antibiotics compared with planktoniccells, as demonstrated by the small amplitude of the Ramanpeaks for planktonic cells compared with sessile cells(Figures 3b and 5). Diallyl sulphide treatment resulted in similarchanges in sessile and planktonic cells; Table 2 depicts Ramanband variations from treatment and suggests that diallylsulphide kills C. jejuni sessile and planktonic cells efficiently.

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Figure 5. Interaction mode between C. jejuni sessile cells with antimicrobial agents. Shown are the second derivative transformations of Ramanspectral features to illustrate variations of untreated C. jejuni sessile cells (black line) and cells treated with 100 mg/L ciprofloxacin (blue line),100 mg/L erythromycin (green line) and 0.1 mg/L diallyl sulphide (red line) for 12 h in a microaerobic environment at 378C. Biofilm EPS waseliminated at this timepoint.

Table 2. Assignment of significant (P,0.05) Raman band variations ofC. jejuni sessile cells treated with 0.1 mg/L diallyl sulphide for 12 h in amicroaerobic environment at 378C

Raman frequency(cm21) Assignment

524 S-S disulphide stretching in proteins540 y (S-S) trans-gauche-trans (amino acid cysteine)920 C-C stretching of proline ring1093 symmetric PO2 stretching vibration of DNA

backbone1131 fatty acid1558 tryptophan1600 amide I


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Furthermore, sessile C. jejuni cells in the untreated biofilm(Figure 5, black lines) showed different Raman spectral featurescompared with their planktonic counterparts (Figure 3b, blacklines). This was in agreement with previous studies showing thatthe physiological condition and chemical composition of sessilecells grown in a biofilm are different from those of planktoniccells due to nutrient limitations, changes in metabolic activityand differences in the localized chemical microenvironment.7,11 –

13 Quiles et al.49 used FT-IR spectroscopy to monitor the spectralvariations of Pseudomonas fluorescens from the planktonic stateto the nascent biofilm state and observed an increase in the con-centration of polysaccharides and proteins during biofilm forma-tion. We observed a similar phenomenon (Figure S4). The biofilmshowed recalcitrance against ciprofloxacin and erythromycin forthe first 5–7 h of antibiotic treatment (Figure 4). Reduced metabol-ic activity may result in less susceptibility of sessile cells to anti-microbial agents, which may explain the differences in bandvariations of antibiotic-treated sessile cells in the biofilm comparedwith planktonic cells following exposure to antimicrobials(Figures 3 and 5). In addition, the genetic response in sessile cellsmay contribute to protective stress responses,10,11,43 whereasplanktonic cells are readily overwhelmed by a strong antimicrobialchallenge and most die before stress responses can be activated.

EPS composition and biofilm architecture could delay thedelivery of antimicrobial agents to cells within the biofilm(Figure 4), providing sessile cells time for physiologically protectiveadaptations (Figure 5). This would be possible if the bacteria in the

biofilm were already able to adjust to a relatively slowly changingconcentration gradient of nutrients and/or antagonists.43 Inaddition, the transition from exponential to slow or no growth isgenerally accompanied by an increase in resistance to antibio-tics.11,12 Here we demonstrated that the high levels of recalcitranceexhibited by C. jejuni biofilms against antibiotics originated from atwo-phase interplay of delayed transport from EPS and physio-logical adaptation of sessile bacterial cells in biofilm.

Electron microscope examination of C. jejuni planktoniccell and biofilm inactivation by diallyl sulphide

SEM revealed that treatment of the sessile C. jejuni cells in abiofilm with 0.1 mg/L diallyl sulphide for 1 h completelydestroyed the EPS structure and cell membrane integrity, indicat-ing the significant antimicrobial activity of this organosulphurcompound (Figure 6). Furthermore, we observed a clear autodis-persion of biofilm cells during SEM sample preparation, indicatingthe potential of using diallyl sulphide as an antimicrobial agenteither alone or in combination with existing antimicrobialtherapies.

PLSR model for prediction of C. jejuni survival numberin biofilms

A PLSR model using wavenumbers ,1800 cm21 as x and an in-dicator variable (loading plot) as y was performed for both FT-IR

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Figure 6. C. jejuni biofilms were inactivated by diallyl sulphide. SEM images of C. jejuni F38011 biofilm without (a, b and c) and with (d) treatment withdiallyl sulphide (0.1 mg/L) in broth for 1 h in a microaerobic environment at 378C.

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and Raman spectra of C. jejuni biofilms to predict the survivingcell numbers following treatment with antimicrobial agents.The model parameters are summarized in Table 3. Because ofthe limited sample numbers, ‘leave-one-out’ cross-validationwas performed. A good PLSR model should have high valuesfor the correlation coefficient (R) (.0.95) and RPD (.5), andlow values for root mean square error (RMSE) of calibrationand RMSE of cross-validation (,1) for calibration and cross-validation. Furthermore, a reasonable number of latent variables(generally ,10) is desired for the PLSR model to avoid overfit-ting.39,40,44 FT-IR and Raman PLSR models showed promisingresults for predicting C. jejuni cell numbers in biofilms exposedto ciprofloxacin, erythromycin and diallyl sulphide. Both infrared-and Raman-based PLSR models provided similar model behav-iour and prediction ability on the basis of R, RPD and RMSE(Table 3).

In the present study we validated that diallyl sulphideeliminated planktonic cells and sessile cells in biofilms at aconcentration that was at least 100-fold less than used for fluor-oquinolones and macrolides on the basis of molarity. The recal-citrance to the antimicrobial agents was due to the limiteddiffusion caused by the biofilm EPS followed by a differentmode of interaction between the sessile and planktonic cells.Based on our data, diallyl sulphide may be a suitable antimicro-bial agent and useful as a natural food preservative.

AcknowledgementsWe thank Dr Valerie Jean Lynch-Holm, who aided us with the electronmicroscope work in the Franceschi Microscopy and Imaging Center atWashington State University, Pullman. We also gratefully acknowledgethe support of University of Idaho Biological Applications ofNanotechnology (BANTech) Center, Moscow, Idaho.

FundingThis work was supported from funds awarded to M. E. K. from the Nation-al Institutes of Health (R56 AI088518-01A1) and funds awarded toB. A. R. from the National Institute of Food and Agriculture (AFRI2011-68003-20096).

Transparency declarationsNone to declare.

Supplementary dataFigures S1 to S4 and Table S1 are available as Supplementary data at JACOnline (http://jac.oxfordjournals.org/).

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FT-IR-CIP 4.89–8.31 84 8 ≥0.98 ≤0.29 ≥10.92 ≥0.95 ≤0.67 ≥7.92FT-IR-ERY 4.08–8.33 84 8 ≥0.98 ≤0.52 ≥12.39 ≥0.95 ≤0.88 ≥10.09FT-IR-DS 3.87–8.29 36 7 ≥0.97 ≤0.37 ≥14.12 ≥0.96 ≤0.71 ≥11.23Raman-CIP 4.89–8.31 84 8 ≥0.99 ≤0.46 ≥12.43 ≥0.97 ≤0.91 ≥9.47Raman-ERY 4.08–8.33 84 7 ≥0.97 ≤0.31 ≥15.85 ≥0.93 ≤0.76 ≥10.98Raman-DS 3.87–8.29 36 7 ≥0.98 ≤0.26 ≥16.07 ≥0.95 ≤0.65 ≥12.31

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Antimicrobial effect of diallyl sulphide on Campylobacter jejuni biofilms