Antimicrobial effect of diallyl sulphide on Campylobacter jejuni biofilms

Abstract

Objectives

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

Methods

C. jejuni biofilms and planktonic cells were treated with ciprofloxacin, erythromycin and diallyl sulphide 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 at least 100-fold less than used for either ciprofloxacin or erythromycin on the basis of molarity. Distinct cell lysis was observed in diallyl sulphide-treated planktonic cells using immunoblot analysis and was confirmed by a rapid decrease in cellular ATP. Two phases of C. jejuni biofilm recalcitrance modes against ciprofloxacin and erythromycin were validated using vibrational spectroscopies: (i) an initial hindered adsorption into biofilm extracellular polymeric substance (EPS) and delivery of antibiotics to sessile cells within biofilms; and (ii) a different interaction between sessile cells in a biofilm compared with their planktonic counterparts. Diallyl sulphide destroyed the EPS structure of the C. jejuni biofilm, after which the sessile cells were killed in a similar 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 may have applications for reducing the prevalence of this microbe in foods, biofilm reduction and, potentially, as an alternative chemotherapeutic agent for multidrug-resistant bacterial strains.

Introduction

Campylobacter jejuni is a leading cause of bacterial gastrointestinal disease worldwide. C. jejuni-mediated disease (campylobacteriosis) is normally self-limiting, but in some instances is associated with severe enteritis, septicaemia and a higher incidence of Guillain–Barré syndrome.¹ In severe cases of campylobacteriosis, erythromycin (a macrolide) or ciprofloxacin (a fluoroquinolone) are commonly used for treatment.² However, fluoroquinolone resistance is common amongst C. jejuni strains and macrolide resistance in strains recovered from clinical isolates has appeared in recent years.^2,3 Only through the development of new antimicrobial agents will it be possible to treat the growing number of multidrug-resistant bacteria.⁴

C. jejuni may form a monospecies biofilm.^5–7 Adherent cells are embedded within an extracellular polymeric substance (EPS) composed of polysaccharides, proteins, nucleic acids, lipids and humic-like substances. The EPS matrix mediates cell/cell communications such as quorum sensing, maintains biofilm hydration and protects microorganisms against environmental stresses, including antibiotic treatments.⁸ The chemical composition and structure of EPS varies markedly, depending upon several factors: cell species, metabolic activity, nutrient availability, biofilm maturity level and physicochemical conditions.⁹

In situ studies indicate that bacteria in biofilms are as much as 1000 times more resistant to antibiotics than planktonic cells.^10–12 The factors contributing to antibiotic resistance include altered physiological growth state and altered microenvironments that impede penetration of the antibiotic into the biofilm.^13,14

New spectroscopic techniques have made it possible to study the properties of microbial biofilms without the confounding effects introduced by staining techniques. For example, traditional fluorescence-induced confocal laser scanning microscopy (CLSM) requires staining. Coupling either Fourier transform infrared (FT-IR) spectroscopy or Raman spectroscopy with confocal microscopy can provide detailed chemical information about microbial cells and complex biofilm matrices without staining. These two spectroscopic methods provide complementary analytical techniques using ‘whole-organism fingerprint’ spectral features of bacteria with spatial resolution in the micrometre range, enabling correlations between optical and chemical images.^15–17 The biochemical properties of healthy and injured microbial cells have been studied by infrared and Raman spectroscopy,^18–25 but there have been few studies investigating biofilm properties.^26–31 Recently, a number of studies have been conducted using vibrational spectroscopy to monitor the inactivation of planktonic bacteria exposed to antibiotics.^4,32–38

Recent studies indicate that plants, specifically Allium spp., contain antimicrobial agents such as diallyl sulphide that are highly effective against major foodborne pathogens.^39,40 We hypothesized that diallyl sulphide might be more effective in inactivating bacterial biofilms than erythromycin or ciprofloxacin based on its ability to freely penetrate the phospholipid bilayers of bacterial cell walls.^39,40 The objectives of this study were to compare systematically the effectiveness of diallyl sulphide with antibiotics commonly used to treat campylobacteriosis. The novelty of this study is that researchers have not examined the antimicrobial activity of diallyl sulphide against any type of bacterial biofilms, including C. jejuni. Moreover, we used biophysical and biochemical techniques to investigate the differences in antimicrobial mechanisms of diallyl sulphide and antibiotics against C. jejuni biofilms. We show for the first time that the antimicrobial activity of diallyl sulphide against C. jejuni planktonic cells and biofilms is much greater than that of selective antibiotics. In addition, we are the first to use vibrational spectroscopy to validate that C. jejuni planktonic cells have a different interaction mode with antibiotics compared with their sessile cell counterparts after biofilm EPS has been destroyed. Planktonic cells and sessile cells have a similar mode of susceptibility to diallyl sulphide, and it was much easier to destroy biofilm EPS with diallyl sulphide than with antibiotics.

Materials and methods

Chemicals and reagents

Ciprofloxacin (purity 98%), erythromycin (purity 95%) and diallyl sulphide (purity 98%) were purchased from Sigma. The purity and stability of diallyl sulphide were determined using methods described in a previous study.³⁹

Bacterial strains and culture methods

Campylobacter jejuni strains F38011 and NCTC 11168 were used in this study. Bacterial cells were cultured either on Mueller–Hinton agar plates supplemented with 5% citrated bovine blood (MHB) or in Mueller–Hinton broth with constant shaking. C. jejuni were incubated in a microaerobic environment (85% N₂, 10% CO₂, 5% O₂) at 37°C, and were passaged every 48 h.

Biofilm formation

Overnight cultures of C. jejuni isolates were diluted to an OD₅₄₀ of 0.03 (∼10⁷ cfu/mL) and 100 μL was added to the surface of a sterile nitrocellulose membrane (0.45 mm pore size, 47 mm diameter; Sartorius Stedim-type filters). The membranes were placed onto agar plates and incubated in a microaerobic environment at 37°C. The membranes were aseptically transferred to new agar plates every 24 h for up to 3 days to form discernible and uniform C. jejuni biofilms with a surface area of ∼1 cm².

Antimicrobial treatments with diallyl sulphide and selected antibiotics

Selected antibiotics [100 mg/L (equivalent to 300 μM) ciprofloxacin and 100 mg/L (equivalent to 136 μM) erythromycin] and/or diallyl sulphide (0.1 mg/L, equivalent to 1 μM) were added to 5 mL bacterial culture grown overnight (∼10⁹ cfu/mL) and incubated for different times (0, 1, 3, 5, 8, 10, 12 and 24 h). The concentration of antibiotics used was based on previous studies.⁴¹ At each sampling time, the bacterial suspensions were serially diluted and spread onto the surface of MHB for enumeration of viable bacteria. For the biofilm experiments, each biofilm was treated with the same antimicrobial agents as mentioned above. Bacterial biofilms coated on nitrocellulose membrane were aseptically removed from MHB agar plates, then placed into 20 mL of MH broth with the same selective concentration of antimicrobial agent (100 mg/L ciprofloxacin, 100 mg/L erythromycin or 0.1 mg/L diallyl sulphide) and incubated with gentle shaking in a microaerobic environment at 37°C. Untreated bacterial biofilms maintained intact EPS and viable sessile cell numbers within biofilms during culture for 24 h. At each sampling time (0, 1, 3, 5, 7, 12 and 24 h), the recovered biofilms were rinsed twice 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 not shown). Following incubation, the trypsin solution was collected, serially diluted and spread onto the surface of MHB for enumeration of viable bacteria.

The MICs of ciprofloxacin, erythromycin and diallyl sulphide against C. jejuni planktonic cells were determined using a broth microdilution method. Briefly, serial 2-fold dilutions of antimicrobials were prepared in a 96-well microtitre plate using MH broth. Freshly grown bacterial cells at log phase were inoculated into each well to give a final concentration of ∼10⁵ cfu/mL. After the inocula had been incubated in a microaerobic environment for 24 h at 37°C, the growth of the bacteria in each well was determined by measuring the absorbance at OD₅₄₀ and compared with the growth of the positive control (a well containing no antimicrobials). The MIC was recorded as the lowest concentration of antimicrobial that completely inhibited bacterial cell growth.

Determination of cytosolic ATP

The ATP Bioluminescence Assay kit CLS II (Roche) was used according to the manufacturer's recommendations to analyse the influence of diallyl sulphide, ciprofloxacin and erythromycin on C. jejuni intracellular ATP levels. Bacterial samples (2 mL, ∼10⁹ cfu/mL) were diluted with deionized water to an appropriate ATP concentration (10⁻⁵ to 10⁻¹⁰ M ATP). Then, 50 μL of each sample was transferred to a microtitre plate that was kept on ice until measurement. Luciferase reagent (50 μL) was added and luminescence read with a VICTOR™ X5 Multilabel Plate Reader (PerkinElmer) at 20°C.

SDS–PAGE and immunoblot analysis

C. jejuni F38011 and NCTC 11168 wild-type strains (∼10⁹ cfu/mL) were treated with 0.1 mg/L diallyl sulphide. Supernatants from these cultures and from untreated controls were obtained following low-speed centrifugation (6000 g) and analysed by SDS–PAGE using standard procedures. For immunoblot analysis, the membranes were incubated with a rabbit anti-C. jejuni polyclonal serum prepared in PBS/Tween [20 mM sodium phosphate and 150 mM sodium chloride, pH 7.5, containing 0.01% (v/v) Tween 20] with 9% non-fat dry milk. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (whole molecule) diluted 1 : 1000 in PBS/Tween and developed 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 untreated and treated with 0.1 mg/L diallyl sulphide for 1 h in a microaerobic environment at 37°C. The C. jejuni biofilm was fixed with 2% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) overnight at 4°C. The fixed biofilms were then washed with phosphate buffer, post-fixed in 1% osmium tetroxide for 2 h at 20°C and rinsed twice with 0.1 M phosphate buffer prior to dehydration in an ethanol series. They were then incubated with 100% hexamethyldisilazane (HMDS) overnight and air dried. These prepared biofilms were mounted onto SEM stubs and sputter coated with a thin layer of gold. The coated samples were observed under an FEI Quanta 200F scanning electron microscope using an accelerating voltage of 30 kV.

FT-IR spectroscopy analysis

At 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 at 20°C. The supernatant was discarded and the pellet resuspended in 20 mL PBS to remove components of the medium. Then, 20 mL was filtered through an aluminium oxide membrane filter (0.2 μm pore size, 25 mm OD) (Anodisc) using a Whatman vacuum glass membrane filter holder to recover bacterial cells. The Anodisc filters were air-dried under laminar flow at 20°C for 60 min. For the biofilm experiment, the nitrocellulose membrane with a biofilm was removed from the agar plate on which it was cultured and placed directly on the diamond crystal cell of a Nicolet 380 FT-IR spectrophotometer (Thermo Electron) for spectral measurement. In agreement with previous work, the nitrocellulose membrane did not interfere with the collection of high-quality spectra.⁴² FT-IR spectroscopy was used to collect sample spectral features for each sample in triplicate, as described in previous studies.^39,40

Raman spectroscopic analysis

Klarite™ (Renishaw Diagnostics) surface-enhanced Raman scattering (SERS)-active substrates were used for planktonic bacterial samples to enhance the intensities of Raman signals. Untreated and treated planktonic C. jejuni cells (10 μL) were deposited onto the substrate and Raman measurements were taken after drying for 1 h under a fume hood at 20°C. For the biofilm experiments, the nitrocellulose membranes coated with the bacterial biofilms were placed directly under a microscope for focus adjustment and employed for Raman spectral collection.

Raman spectroscopic analysis was performed using a WITec alpha300 Raman microscope (WITec, Ulm, Germany) equipped with a UHTS-300 spectrometer and a 532 nm laser delivering ∼2 mW incident laser power on the planktonic bacterial samples and ∼0.2 mW incident laser 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 was 16 × 16 μm. WITec Control v1.5 software (WITec, Ulm, Germany) was employed for instrumental control and data collection. Raman spectra of each planktonic bacterial and/or biofilm sample were collected over a simultaneous wavenumber shift range of 3700–200 cm⁻¹ in extended mode. For measurement at a single location, each full spectral measurement was conducted with a 3 s integration time with 20 spectral accumulations (total integration time 60 s). Eight spectra were collected for each sample in triplicate.

In situ Raman mapping analysis

Full area raster scans were performed to create Raman maps, with single-spectrum integration times of 3 s, saving 600 spectra acquired over 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 xyz motorized stage was employed during Raman map generation. Subsequent band analysis for the collected spectra was applied to create intensity-correlated maps for relevant Raman bands. Thus, chemical images corresponding to optical images of the biofilm were generated based upon the area beneath the baseline-corrected specific bands. An estimate of the distribution and concentration of biochemical constituents within a section of the biofilm could be determined subsequently (N = 15).

Spectroscopic-based chemometric analysis

ASCII data pertaining to individual Raman spectra were exported from WITec Project software. Infrared and Raman spectra were initially preprocessed using OMNIC (Thermo Electron). The spectra were subtracted from relative background if necessary. Automatic baseline correction was then employed to flatten the baseline, followed by smoothing using a Gaussian function with a bandwidth of 9.643 cm⁻¹. The height and area of vibrational spectral bands were measured and calculated using OMNIC and Matlab. Second-derivative transforms of vibrational spectra using a nine-point Savitzky–Golay filter and wavelet transforms with a scale of 7 were performed in Matlab. The reproducibility of infrared and Raman spectra was determined by calculating D_y1y2 as described in previous studies.^39,40 Partial least squares regression (PLSR) models were established based upon processed vibrational spectra and employed for quantitative analyses in Matlab. A total of four spectra of each sample were used to establish the calibration model (N = 30). A ‘leave-one-out’ cross-validation was employed to study the predictive power of the PLSR chemometric model by removing vibrational spectra for one sample from the dataset and applying a calibration to the remaining samples.⁴⁰ The overall suitability of the developed chemometric models for predicting live C. jejuni numbers in the biofilm was evaluated using residual prediction deviation (RPD) values and other parameters described in previous studies.^25,39,40

Statistical analysis

Each experiment was independently repeated a minimum of three times to ensure reproducibility. The results were expressed as the mean of three independent replicates ± standard deviation. The significance of differences between band heights of raw and second derivative transformed spectra was determined using one-way analysis of variance following t-test in Matlab to determine critical variations of chemical components of samples. Differences were considered significant at P < 0.05.

Results and discussion

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

In the current study, we used C. jejuni planktonic cells as the control to determine and compare the effectiveness of diallyl sulphide and two selective antibiotics, ciprofloxacin and erythromycin, in inactivating C. jejuni biofilms. In addition, we hypothesized that C. jejuni planktonic cells and sessile cells in biofilms have different interaction modes and susceptibility mechanisms to treatment with diallyl sulphide and antibiotics. We used planktonic cells as a control throughout our experiments to investigate the antimicrobial mechanism of these antimicrobials. Two strains of C. jejuni were used, strain F38011 (a human clinical isolate) and NCTC 11168 (a sequenced strain). The MIC values of ciprofloxacin for these strains were identical (16 mg/L), but the strains differed in their susceptibility to erythromycin. The MIC for C. jejuni F38011 was 8 mg/L and that for strain NCTC 11168 was 4 mg/L. An MIC of 0.04 mg/L was established for diallyl sulphide. The survival of planktonic and sessile cells treated with these antimicrobial agents, at concentrations exceeding these MICs, was examined over a 24 h time period (Figure 1). Diallyl sulphide eliminated planktonic cells and sessile cells of both strains much faster than the antibiotics did, at a concentration that was 136- to 300-fold less than used for erythromycin or ciprofloxacin, respectively.

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 dotted lines) in a microaerobic environment at 37°C. Panels (a) and (b) show data for planktonic cells and panels (c) and (d) show data for sessile cells in biofilm. Strain C. jejuni NCTC 11168 is shown in panels (a) and (c) and strain F38011 is shown in panels (b) and (d).

The survival of the C. jejuni F38011 and NCTC 11168 biofilms treated with antimicrobial agents for 24 h are also shown in Figure 1. Sessile cells exhibited greater resistance to treatment with ciprofloxacin and erythromycin than planktonic cells, demonstrating the recalcitrant properties of the biofilm (Figure 1). Diallyl sulphide treatment totally inactivated the cells within the biofilm within 5 h compared with >24 h required for ciprofloxacin and erythromycin, as determined by the number of viable bacteria recovered following treatment. This is the first time diallyl sulphide has been shown to have a significantly higher antimicrobial effect against bacterial biofilms compared with commonly used antibiotics. Furthermore, these data also suggest that the interaction mode of antimicrobial action of diallyl sulphide is different from that of the two antibiotics.

Inhibition of cellular metabolism—cytosolic ATP levels

To address the antimicrobial mechanism of diallyl sulphide against C. jejuni, we first studied its antimicrobial mechanism against planktonic bacterial cells. Cellular ATP levels correlate with cell viability. After a loss of membrane integrity, cells lose the capacity to synthesize ATP, and endogenous ATPases destroy any remaining ATP; thus, ATP levels drop precipitously. ATP levels of C. jejuni treated with 0.1 mg/L diallyl sulphide decreased markedly after 1.5 h of treatment (Figure S1, available as Supplementary data at JAC Online). However, the ATP levels of C. jejuni treated with either 100 mg/L ciprofloxacin or 100 mg/L erythromycin remained stable following a 12 h treatment (Figure S2, available as Supplementary data at JAC Online). The initial increase in ATP levels observed in C. jejuni treated with ciprofloxacin is possibly due to the inhibition of cell division and the resulting increase in the volume of individual cells. In addition, neither ciprofloxacin nor erythromycin is known to compromise bacterial membrane integrity. Thus, a drop in ATP is not expected for these compounds until the cells are no longer viable. Taken together, these results demonstrate that diallyl sulphide decreases cytosolic ATP and subsequently inhibits cellular metabolism and inactivates bacterial cells compared with ciprofloxacin and erythromycin.

Planktonic cell lysis by diallyl sulphide is validated by immunoblot analysis

There are several possible mechanisms to explain the decline in bacterial viability observed upon treatment with diallyl sulphide, including bacterial cell lysis, inactivation of key metabolic proteins and inhibition of protein synthesis. Treatment of planktonic cells with 0.1 mg/L diallyl sulphide resulted in cell lysis or leakage, as judged by immunoblot analysis (Figure S3, available as Supplementary data at JAC Online).

Infrared and Raman spectral features of C. jejuni biofilm

Infrared and Raman spectral features of C. jejuni planktonic cells have been reported previously.^18,39 Figure 2 shows an optical microscope image and typical Raman spectra of a single-species biofilm (C. jejuni NCTC 11168) obtained at a 532 nm excitation wavenumber and a 60 s integration time. Spectral features associated with polysaccharides, nucleic acids and proteins are apparent in the Raman spectra for these biofilms and are impacted by antimicrobial treatment. The methods employed here provided greater detail of the biochemical features of bacterial biofilms than recent studies in which multispecies biofilms had been characterized using surface-enhanced Raman spectroscopy.^26,27 Raman and SERS spectral features reflect similar chemical properties, but do not necessarily coincide due to chemical enhancemment effects that may cause band shifts. Interaction between specific analytes in biofilm and SERS active surface (hot spots) may also cause variations from band shifts.^20,22,27 The FT-IR and Raman band assignments for C. jejuni biofilm are summarized in Table S1 (available as Supplementary data at JAC Online) and a comparison of FT-IR spectral features for the C. jejuni biofilm and planktonic cell is shown in Figure S4 (available as Supplementary data at JAC Online). The chemical composition of biofilm could be determined using FT-IR and Raman spectroscopies. In addition, we demonstrated that the protein and polysaccharide compositions of C. jejuni planktonic cells and biofilms were different in the present study.

Figure 2.

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

Reproducibility of Raman mapping and vibrational spectroscopy for C. jejuni biofilm

Spectral reproducibility is critical and is the precondition for chemometric statistical analyses, such as second-derivative transformation and its comparison. The distribution of chemical components on biofilms will also affect spectral reproducibility and this distribution can be determined using spectroscopy-based mapping techniques. Thus, we determined the different chemical components of the C. jejuni biofilm matrices using confocal Raman mapping (Figure 2). The spectra from a specific biofilm area were obtained by raster scans. Raster scanning is a technique for capturing a video rectangular pattern of an image line by line. Raman maps of the relevant band intensities were calculated and correlated with the regions on the optical images of the biofilm from which they were taken. Figure 2(b) shows the Raman map of the band intensity at 868 cm⁻¹ collected from an area of 10 × 10 μm (see black frame in Figure 2a). This Raman map shows the distribution of monosaccharide/polysaccharide in the upper biofilm layer. Previous work has demonstrated that the distribution of proteins and polysaccharides in the biofilm can be mapped at other wavelength regions with marker bands selected for the relevant biological substances.²⁷ However, being able to compare experimental results is dependent upon being able to obtain a reliable estimate of reproducibility.

The reproducibility of vibrational spectra from independent experiments and various sample locations was calculated as D_y1y2 values. Mean D values between 7 and 10 are considered to be normal when analyzing the first- or second-derivative transforms of spectral features from samples prepared in independent assays.^18,39,40 Others have asserted that D values can be as high as 300 when microorganisms from different genera are compared,¹⁸ but there are no reported studies by other investigators calculating spectral reproducibility for bacterial biofilms. Wavenumber and cultivation time were critical to the reproducibility of biofilm vibrational spectra, the same as for bacterial planktonic cells.^18,39,40 The wavenumber region for Raman spectra of C. jejuni biofilms was from 1800 to 400 cm⁻¹ while the wavenumber region for FT-IR spectra of C. jejuni biofilms ranged from 1800 to 700 cm⁻¹ (‘fingerprint’ region). The D value for Raman spectra was 7.19 ± 1.12 to 9.10 ± 1.56 and the D value for FT-IR spectra was 16.96 ± 2.25 to 21.39 ± 3.92 after 72 h of culture on MHB agar. Both FT-IR and Raman spectra offer high reproducibility, with the reproducibility of Raman spectra being significantly (P < 0.05) greater than that for FT-IR spectra. While physiological heterogeneity is common in biofilms,⁴³ we found that the heterogeneity did not significantly affect (P > 0.05) vibrational spectral reproducibility. Collectively, our data show high spectral reproducibility and this is critical for the development of reliable chemometric models.⁴⁴

Mode of C. jejuni planktonic cell inactivation by antimicrobial agents

For the second-derivative transformation analyses of FT-IR and Raman spectra, the planktonic cells were treated with ciprofloxacin, erythromycin and diallyl sulphide for 8 h and the bacterial spectral variations compared with one another (Figure 3a and b). For C. jejuni planktonic cells treated with 100 mg/L ciprofloxacin, significant (P < 0.05) band variations were related to DNA/RNA information. Table 1 provides the detailed identification for FT-IR and Raman band variations. Consistent with previous work, we demonstrated from spectral measurement that ciprofloxacin alters DNA structure in C. jejuni planktonic cells.

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 cells treated 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 microaerobic environment at 37°C. (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) for 8 h in a microaerobic environment at 37°C.

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 37°C

Treatment/FT-IR frequency (cm−1)	Assignment	Raman frequency (cm−1)	Assignment
Ciprofloxacin 100 mg/L
800	left-handed helix DNA	788	C5-O-P-O-C3 phosphodiester bands in DNA
1084	phosphate/sugar backbone of nucleic acids	828	O-P-O stretch DNA
1220	stretching phosphate antisymmetric vibration in B-form DNA	1093	symmetric PO2 stretching vibration of the DNA backbone
1606	adenine vibration in DNA	1243	phosphodiester groups of nucleic acids
		1458	nucleic acid mode
Erythromycin 100 mg/L
1400	symmetric CH3 bending modes of methyl groups of proteins	760	ring breathing tryptophan
1455	symmetric bending modes of methyl groups in skeletal proteins	1004	phenylalanine
1655	amide I (α-helix)	1260	amide III vibration mode of structural proteins
Diallyl sulphide 0.1 mg/L
916	phosphodiester	524	S-S disulphide stretching in proteins
991	phosphodiester	540	υ(S-S) trans-gauche-trans (amino acid cysteine)
1220	stretching phosphate antisymmetric vibration in B-form DNA	760	ring breathing tryptophan
1515	amide II	1131	fatty acid
1545	amide II	1323	guanine
1637	amide I	1470	C = N stretching
1655	amide I

For C. jejuni planktonic cells treated with 100 mg/L erythromycin, significant (P < 0.05) band variations were related to the secondary structure of proteins. Table 1 provides identification for FT-IR and Raman bands. Spectral variations in secondary structure of protein regions found in this study confirm that bacterial protein synthesis and inhibition of processes critical for cell function or replication are inhibited by macrolides that bind to the 50S subunit of the bacterial 70S rRNA complex.²

For C. jejuni planktonic cells treated with 0.1 mg/L (1 μM) diallyl sulphide, significant (P < 0.05) band variations were observed in the whole fingerprint wavenumber regions for both the FT-IR and Raman spectra. Table 1 provides the identification for FT-IR and Raman band variations. The spectral variations in the sulphur region determined by Raman spectroscopy are in agreement with previous studies using higher concentrations of diallyl sulphide (5, 10 and 20 μM) for different treatment times,^39,40 and suggest that inhibition of certain thiol-containing enzymes/proteins in C. jejuni by the rapid reaction of diallyl sulphide with thiol groups may be an important mechanism for antimcrobial activity. The transmembrane transfer of sulphur-containing compounds into C. jejuni could be monitored using Raman spectroscopy, but not FT-IR spectroscopy, emphasizing the importance of using complementary vibrational spectroscopies at the same time for studies of bacterial resistance and inactivation against antimicrobial agents.

C. jejuni biofilm EPS recalcitrance mode against antimicrobial agents

Biofilms show greater recalcitrance towards antimicrobial agents when compared with planktonic cells.³⁴ Recalcitrance is reflected in the infrared spectral features of C. jejuni biofilm that were different from those of planktonic cells at three wavenumbers (Figure S4). Specifically, wavenumber differences were observed in the amide III band components of proteins (band 1280 cm⁻¹), the ring structure of polysaccharides (band 1162 cm⁻¹) and the C₂ endo conformation of polysaccharides (band 829 cm⁻¹).¹⁶ These three bands reflect the properties of the major components of biofilm EPS^5,8 and were selected to monitor biofilm EPS variations in response to antibiotic and diallyl sulphide treatment.

We observed no structural difference in the C. jejuni biofilm EPS after ciprofloxacin (100 mg/L) treatment for approximately 7 h and erythromycin (100 mg/L) treatment for ∼5 h. However, the EPS was totally destroyed by diallyl sulphide (0.1 mg/L) treatment within 1 h (Figure 4). FT-IR spectral variations at 1280, 1162 and 829 cm⁻¹ indicated recalcitrance of the biofilm EPS structure to ciprofloxacin and erythromycin, showing that EPS provides a physical or chemical diffusion barrier and alters adsorption properties to antimicrobial penetration into the biofilm, preventing the access of antibiotics to the embedded sessile cells.^11–14 Reaction of antibiotics with or adsorption to biofilm components can limit transport into the cells in a biofilm.^14,45 Suci et al.³² used FT-IR spectroscopy to monitor the impeded transport of ciprofloxacin to the biofilm–substratum interface and direct interaction of biofilm components with the antibiotic in a flowing system. Similarly, prevention of diffusion of piperacillin into Pseudomonas aeruginosa biofilms has been observed.⁴⁶ In addition, increasing the size of the hydrophobic side chains of selected quaternary ammonium compounds reduced the susceptibility of Staphylococcus aureus treated with antibiotics when these bacteria were embedded in a hydrophobic EPS matrix.⁴⁷ Biofilm EPS is not impenetrable to antibiotics, but transport is impeded.¹² We demonstrated that the biofilm EPS was intact during antibiotic treatment during the first several hours and that there was a transport delay of antibiotic compounds to the sessile bacterial cells within the biofilm. The biofilm EPS began to decay after this initial recalcitrance (Figure 4). However, given the biofilm survival curves shown in Figure 1, other mechanisms must also be acting to support biofilm cell survival besides diffusion limitations.

Figure 4.

Treatment of C. jejuni biofilm with diallyl sulphide causes greater alterations in EPS proteins and polysaccharides than ciprofloxacin and erythromycin as evidenced by FT-IR spectroscopic analysis. Shown are the variations of specific FT-IR bands related to biofilm untreated EPS (black lines) and EPS treated with 100 mg/L ciprofloxacin (black dotted lines), 100 mg/L erythromycin (grey lines) and 0.1 mg/L diallyl sulphide (grey dotted lines). (a) 1280 cm⁻¹, amide III band components of proteins. (b) 1162 cm⁻¹, ring structure of polysaccharides. (c) 829 cm⁻¹, C₂ endo conformation of polysaccharide.

The biofilm structure was destroyed by 0.1 mg/L diallyl sulphide within 1 h, explaining why diallyl sulphide had a more powerful antimicrobial effect on C. jejuni biofilm compared with the antibiotics tested at the same concentrations. It is known that organosulphur compounds can freely penetrate through the phospholipid bilayers of bacterial cell walls⁴⁸ and cause planktonic cell lysis (Figure S3). We hypothesize that organosulphur compounds destroy the biofilm EPS structure more easily than erythromycin and ciprofloxacin because diallyl sulphide is more polar, smaller and hydrophilic. This biological profile provides a faster penetration of diallyl sulphide through biofilm EPS to sessile cells. Furthermore, disaggregation of a biofilm will increase the effectiveness of an antimicrobial agent.¹¹ What remains to be determined is whether diallyl sulphide causes the EPS to detach from the biofilm structure or whether the EPS is eliminated by other means.

C. jejuni sessile cells in biofilm had a different interaction mode with antimicrobial agents compared with planktonic cells

C. jejuni biofilm EPS was totally destroyed after 12 h of treatment with antimicrobial agents (Figure 4) and subsequently provided sessile cells with little protection. Significant (P < 0.05) band variations in Raman spectra between treated and untreated sessile cells were determined (Figure 5). Ciprofloxacin- and erythromycin-treated sessile cells show band variations at the same three wavenumbers: ring breathing tryptophans (760 cm⁻¹),¹⁶ CH₃CH₂ wagging mode in purine bases of DNA (1324 cm⁻¹)¹⁷ and bending modes of methyl groups (1401 cm⁻¹).¹⁷ Compared with Figure 3(b), sessile cells in a biofilm had a different interaction mode with antibiotics compared with planktonic cells, as reflected in band variations at different wavenumbers. In addition, sessile cells in the biofilm were more resistant to antibiotics compared with planktonic cells, as demonstrated by the small amplitude of the Raman peaks for planktonic cells compared with sessile cells (Figures 3b and 5). Diallyl sulphide treatment resulted in similar changes in sessile and planktonic cells; Table 2 depicts Raman band variations from treatment and suggests that diallyl sulphide kills C. jejuni sessile and planktonic cells efficiently.

Figure 5.

Interaction mode between C. jejuni sessile cells with antimicrobial agents. Shown are the second derivative transformations of Raman spectral 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 37°C. Biofilm EPS was eliminated at this timepoint.

Table 2.

Assignment of significant (P < 0.05) Raman band variations of C. jejuni sessile cells treated with 0.1 mg/L diallyl sulphide for 12 h in a microaerobic environment at 37°C

Raman frequency (cm−1)	Assignment
524	S-S disulphide stretching in proteins
540	υ(S-S) trans-gauche-trans (amino acid cysteine)
920	C-C stretching of proline ring
1093	symmetric PO2 stretching vibration of DNA backbone
1131	fatty acid
1558	tryptophan
1600	amide I

Furthermore, sessile C. jejuni cells in the untreated biofilm (Figure 5, black lines) showed different Raman spectral features compared with their planktonic counterparts (Figure 3b, black lines). This was in agreement with previous studies showing that the physiological condition and chemical composition of sessile cells grown in a biofilm are different from those of planktonic cells due to nutrient limitations, changes in metabolic activity and differences in the localized chemical microenvironment.^7,11–13 Quiles et al.⁴⁹ used FT-IR spectroscopy to monitor the spectral variations of Pseudomonas fluorescens from the planktonic state to the nascent biofilm state and observed an increase in the concentration of polysaccharides and proteins during biofilm formation. We observed a similar phenomenon (Figure S4). The biofilm showed recalcitrance against ciprofloxacin and erythromycin for the first 5–7 h of antibiotic treatment (Figure 4). Reduced metabolic activity may result in less susceptibility of sessile cells to antimicrobial agents, which may explain the differences in band variations of antibiotic-treated sessile cells in the biofilm compared with planktonic cells following exposure to antimicrobials (Figures 3 and 5). In addition, the genetic response in sessile cells may contribute to protective stress responses,^10,11,43 whereas planktonic cells are readily overwhelmed by a strong antimicrobial challenge and most die before stress responses can be activated.

EPS composition and biofilm architecture could delay the delivery of antimicrobial agents to cells within the biofilm (Figure 4), providing sessile cells time for physiologically protective adaptations (Figure 5). This would be possible if the bacteria in the biofilm were already able to adjust to a relatively slowly changing concentration gradient of nutrients and/or antagonists.⁴³ In addition, the transition from exponential to slow or no growth is generally accompanied by an increase in resistance to antibiotics.^11,12 Here we demonstrated that the high levels of recalcitrance exhibited by C. jejuni biofilms against antibiotics originated from a two-phase interplay of delayed transport from EPS and physiological adaptation of sessile bacterial cells in biofilm.

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

SEM revealed that treatment of the sessile C. jejuni cells in a biofilm with 0.1 mg/L diallyl sulphide for 1 h completely destroyed the EPS structure and cell membrane integrity, indicating the significant antimicrobial activity of this organosulphur compound (Figure 6). Furthermore, we observed a clear autodispersion of biofilm cells during SEM sample preparation, indicating the potential of using diallyl sulphide as an antimicrobial agent either alone or in combination with existing antimicrobial therapies.

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 with diallyl sulphide (0.1 mg/L) in broth for 1 h in a microaerobic environment at 37°C.

PLSR model for prediction of C. jejuni survival number in biofilms

A PLSR model using wavenumbers <1800 cm⁻¹ as x and an indicator variable (loading plot) as y was performed for both FT-IR and Raman spectra of C. jejuni biofilms to predict the surviving cell numbers following treatment with antimicrobial agents. The model parameters are summarized in Table 3. Because of the limited sample numbers, ‘leave-one-out’ cross-validation was performed. A good PLSR model should have high values for the correlation coefficient (R) (>0.95) and RPD (>5), and low values for root mean square error (RMSE) of calibration and 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 overfitting.^39,40,44 FT-IR and Raman PLSR models showed promising results for predicting C. jejuni cell numbers in biofilms exposed to ciprofloxacin, erythromycin and diallyl sulphide. Both infrared- and Raman-based PLSR models provided similar model behaviour and prediction ability on the basis of R, RPD and RMSE (Table 3).

Table 3.

PLSR models for quantification of viable C. jejuni cells in biofilm treated with 100 mg/L ciprofloxacin, 100 mg/L erythromycin or 0.1 mg/L diallyl sulphide in a microaerobic environment at 37°C (N = 30)

Spectraa	Range (cfu/cm2)	No. of samples	No. of latent variables	R cal	RMSE cal	RPD cal	R cv	RMSE cv	RPD cv
FT-IR-CIP	4.89–8.31	84	8	≥0.98	≤0.29	≥10.92	≥0.95	≤0.67	≥7.92
FT-IR-ERY	4.08–8.33	84	8	≥0.98	≤0.52	≥12.39	≥0.95	≤0.88	≥10.09
FT-IR-DS	3.87–8.29	36	7	≥0.97	≤0.37	≥14.12	≥0.96	≤0.71	≥11.23
Raman-CIP	4.89–8.31	84	8	≥0.99	≤0.46	≥12.43	≥0.97	≤0.91	≥9.47
Raman-ERY	4.08–8.33	84	7	≥0.97	≤0.31	≥15.85	≥0.93	≤0.76	≥10.98
Raman-DS	3.87–8.29	36	7	≥0.98	≤0.26	≥16.07	≥0.95	≤0.65	≥12.31

cal, calibration; cv, cross-validation; CIP, ciprofloxacin; ERY, erythromycin; DS, diallyl sulphide.

^aFor FT-IR spectroscopy, a wavenumber from 1800 to 700 cm⁻¹ was used for model analyses; for Raman spectroscopy, a wavenumber from 1800 to 400 cm⁻¹ was used for model analyses.

In the present study we validated that diallyl sulphide eliminated planktonic cells and sessile cells in biofilms at a concentration that was at least 100-fold less than used for fluoroquinolones and macrolides on the basis of molarity. The recalcitrance to the antimicrobial agents was due to the limited diffusion caused by the biofilm EPS followed by a different mode of interaction between the sessile and planktonic cells. Based on our data, diallyl sulphide may be a suitable antimicrobial agent and useful as a natural food preservative.

Funding

This work was supported from funds awarded to M. E. K. from the National Institutes of Health (R56 AI088518-01A1) and funds awarded to B. A. R. from the National Institute of Food and Agriculture (AFRI 2011-68003-20096).

Transparency declarations

None to declare.

Supplementary data

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