Introduction

Campylobacter jejuni is an important food-borne pathogen that causes gastrointestinal illness worldwide. Contaminated chicken and other poultry are the main sources of infection in humans (Ganan et al. 2012). Many chickens raised for food are contaminated with C. jejuni. Therefore, it is essential to reduce the incidence of contamination during food production to reduce subsequent disease (Hald 2010). Campylobacter pathogenesis depends on the expression of virulence factors, several of which control motility and biofilm formation (Svensson et al. 2009). Motility is essential for intestinal colonization and invasion (Guerry 2007). Campylobacter-forming biofilms demonstrate superior resistance to environmental and pharmacological treatments (Gunther and Chen 2009). Due to the increasing incidence of antimicrobial resistance, new strategies are continually being evaluated to control microorganisms without inducing resistance (Steenackers et al. 2010). Such strategies involve finding alternative drugs that selectively inhibit virulence without affecting the planktonic growth of bacteria (Steenackers et al. 2010; Clatworthy et al. 2007).

Most efforts to inhibit the regulation of virulence factor expression have focused on quorum sensing (QS), a complex regulatory process that is dependent on the bacterial cell density. QS is involved in physiological processes, such as biofilm formation, bioluminescence, antibiotic synthesis, and virulence factor expression (Landini et al. 2010). QS systems employ a wide range of signaling molecules. These so-called “autoinducers” regulate changes in gene expression and the subsequent initiation of cooperative bacterial processes that allow pathogenesis (Galloway et al. 2012). Autoinducer 2 (AI-2) is a key molecule to control QS in Campylobacter (Annous et al. 2009; Moorhead and Griffiths 2011).

Various QS inhibitors have been reported. An important class of QS inhibitors includes the brominated furanones produced by the algae Delisea pulchra (Girennavar et al. 2008). These natural furanones interfere with the QS system in several microorganisms (Roy et al.; 2010). Many synthetic furanones appear to be more effective than their natural analogs (Khmel and Metlitskaya 2006). Although furanones have been demonstrated to exhibit differences in efficacy, the antimicrobial efficacy of 2(5H)-furanone is not well known. Moreover, none of these compounds has been investigated in Campylobacter. Epigallocatechin gallate (EGCG), a compound that can be extracted from green tea, exhibits antimicrobial activity and anti-AI-2-mediated QS activity in Escherichia coli (Cho et al. 2010; Lee et al. 2009). This compound acts as a modulator of Campylobacter resistance to macrolide antibiotics (Kurinčič et al. 2012a, b); however, it has not been tested as a QS inhibitor in this microorganism. On the other hand, disinfectants from citrus origin exhibit antibacterial activity against Salmonella and E. coli (Orue et al. 2013) and C. jejuni (Castillo et al. 2014), but no information about the efficacy of isolated compounds on C. jejuni is available.

Because these natural products have been the basis for the development of a broad spectrum of synthetic antibacterial agents (Zang et al. 2009) and C. jejuni is one of the most common food-borne pathogens, the objective of this study was to determine the effects of 2(5H)-furanone, EGCG, and a natural disinfectant on the AI-2-mediated QS system of C. jejuni and the subsequent inhibition of bacterial motility and biofilm formation.

Material and methods

Antimicrobial compounds

2(5H)-Furanone (product no. 283754) and EGCG from green tea (product no. E4268) were obtained from Sigma-Aldrich (St. Louis, MO). A natural disinfectant (Citrol K Ultra®) was obtained from Corpo Citrik SA de CV, Mexico, D.F. (hereinafter, “the disinfectant”) (Orue et al. 2013). Stock concentrations of EGCG, furanone, and the disinfectant were prepared in sterile deionized water.

Bacterial cultures and growth conditions

The following C. jejuni strains were used in this study: NCTC 11168 acquired from ATCC culture collection (Manassas VA, USA.), NADC 5653 (isolated from chicken and kindly provided by Dr. Irene Wesley, USDA-ARS-NADC, Ames, IA, USA), 180ip, and 238ip (both isolated from patients with diarrhea and kindly provided by Dr. Guillermo Ruiz-Palacios from the National Institute of Medical Sciences and Nutrition, Mexico, D.F.). All Campylobacter strains were stored at −80 °C in brain heart infusion (BHI) broth (Difco Laboratories, Sparks, MD, USA) containing 20 % glycerol and supplemented with 0.6 % yeast extract. Fresh cultures were prepared in Mueller-Hinton media (MH) (Difco Laboratories) supplemented with 5 % (v/v) defibrinated horse blood (Castillo et al. 2011) and incubated at 42 °C under microaerobic conditions (10 % CO2) in a Shell Lab CO2 incubator (Cornellius, OR, USA).

For the bioluminescence assay, the Vibrio harveyi reporter strain BB170 phenotype sensor 1−, sensor 2+ (luxN:Tn5) was used. Supernatants of V. harveyi BB152 containing AI-2 (wild type) served as a positive control to measure AI-2 activity. Autoinducer bioassay (AB) or Luria Bertani (LB) medium was used to culture the V. harveyi strains (Lu et al. 2004; Surette and Bassler 1998). These Vibrio strains were acquired from ATCC culture collection (Manassas VA, USA).

Antibacterial assays

The preliminary screening of the antimicrobial properties of compounds was performed by a diffusion technique in agar, as described previously (Castillo et al. 2011). Briefly, petri dishes (150 mm) were filled with 25 mL of MH agar containing 5 % (v/v) defibrinated horse blood. Aliquots (100 μL) of Campylobacter cultures (1 × 106 CFU/mL) were homogeneously spread on the agar surface with a sterile Digralsky spreader. All strains were inoculated separately. Five holes (12 mm in diameter) were made in the seeded agar plate. The holes were then filled with 100 μL of EGCG (1 mg/mL), furanone (10 mg/mL), or disinfectant (0.4 mg/mL). An equivalent volume of water served as negative control. Dishes were incubated microaerobically (10 % CO2 for 48 h at 42 °C). Inhibitory activity was determined by the absence of bacterial growth in the areas surrounding the holes that were filled with each compound. The inhibition zone was measured using a caliper.

Minimal bactericidal concentration (MBC)

The MBC was determined with a dilution method (Sánchez et al. 2010). Each stock compound (20 μL) was placed in 180 μL of MH broth containing 0.6 % yeast extract. Dilutions were prepared in microtiter plates (Corning Costar, Cambridge MA, USA), and final concentrations of compounds ranged from 0.2 μg/mL to 1 mg/mL in a final volume of 200 μL. One microliter from fresh cultures of Campylobacter (1 × 106 CFU/mL) was added to each well. Water or tetracycline (0.1 mg/mL, final concentration, Sigma-Aldrich Co., St. Louis, MO, USA, product no. T-3383), instead of natural compounds, were used as negative and positive controls, respectively (Castillo et al. 2011; Kurincic et al. 2012b). Culture plates were incubated microaerobically (10 % CO2 for 48 h at 42 °C), and bacterial survival was determined by culturing 100 μL of each culture on MH agar containing 5 % (v/v) defibrinated horse blood. The MBC was defined as the lowest concentration of each compound that resulted in no growth of C. jejuni on the MH agar plates. The effects of subinhibitory concentrations of each compound (MBC × 0.25, 0.5, 0.75) on bacterial growth were also determined by colony enumeration in MH agar (Castillo et al. 2011).

Autoinducer bioassay

A bioluminescence assay was used to determine the effect of compounds on AI-2-mediated QS (Surette and Bassler 1998; Lee et al. 2009). Cell-free culture supernatants (CFSNs) of C. jejuni and V. harveyi strain BB152 were prepared as previously described (Cloak et al. 2002). Briefly, C. jejuni strains were grown in Brucella broth at 42 °C for 40 h under microaerobic conditions. Bacteria were removed from the medium by centrifugation at 8,000 g for 15 min, followed by filter sterilization (0.22 μm). CFSNs were stored at −20 °C until use. V. harveyi BB152 was grown overnight in LB Broth at 30 °C with aeration. CFSNs were prepared as described for C. jejuni.

AI-2 activity in CFSNs was measured with the V. harveyi bioluminescence assay (Plummer et al. 2011; Surette and Bassler 1998). Briefly, V. harveyi strain BB170 was grown overnight in LB broth at 30 °C, then diluted 1:5,000 into fresh AB medium to obtain a final concentration of 104 CFU/mL (Soni et al. 2008; Girennavar et al. 2008). Aliquots (90 μL) were added to 96-well microtiter plates, and 10 μL of each CFSN were added. CFSN from the V. harveyi strain BB152 was used as a positive control, whereas the addition of sterile AB medium (with or without 10 μL of LB or Brucella broth) served as the negative control. Luminescence was measured at 30 °C every 20 min over a 12-h period using a Victor X2 2030 multilabel plate reader (Perkin Elmer, Waltham, MA, USA). Results were reported as percentage changes in relative light units (RLUs) after 4 h of incubation, the time when V. harveyi BB170 reaches the exponential phase and maximal autoinducer activity occurs (Cloak et al. 2002; Lu et al. 2004; Lee et al. 2009). Assays were performed in duplicate and replicated three times.

Effects of antimicrobial compounds on AI-2

To test the effects of each compound on AI-2 activity, 5 μL of each CFSN was mixed with 1.5 μL of each compound (MBC × 0.75, final concentration) and 3.5 μL of AB medium. This mixture was added to the autoinducer bioassay (Girennavar et al. 2008). The AI-2 activity was determined by bioluminescence, as described above. These concentrations of the compounds were also tested against growth of V. harveyi BB170 in the autoinducer bioassay to ensure that decrease in bioluminescence was not due to the cell death.

Motility assays

Motility assays were performed by the method of Elvers and Park (2002) and Lee et al. (2009), with minor modifications. Petri dishes were prepared with semisolid MH agar (0.3 % w/v). Subinhibitory concentrations of each compound (MBC × 0.25, 0.5, 0.75) were mixed with the semisolid agar, and 5 μL of the fresh Campylobacter culture (106 UFC/mL) was placed in the center of each plate. Plates were incubated for 48 h at 42 °C under microaerobic conditions. The motility halos were measured with a caliper after 48 h, compared with the control (without compound) and the percent of reduction determined. A nonmotile C. jejuni 180ip strain was used as a negative control.

Biofilm assays

Biofilm assays were performed using the methods described by Møretrø et al. (2003) and Reeser et al. (2007), with minor modifications. Briefly, fresh cultures of C. jejuni strains were adjusted with sterile saline solution (0.85 % p/v) to an A 600 of 0.5 (1 × 106 CFU/mL). Then, 1 μL of the bacterial suspension was used to inoculate 96-well polystyrene plates (Corning Costar, Cambridge MA, USA) containing 100 μL of Brucella broth (Difco) and each compound at a MBC × 0.75. Plates were incubated microaerobically at 42 °C for 72 h. Medium was then removed, and wells washed two times with distilled water, then dried for 30 min at 55 °C.

The wells were stained by adding 100 μL of 0.1 % safranin (Sigma) for 15 min at room temperature. Unbound safranin was removed by two washes with distilled water. The plates were dried at 55 °C for 15 min, and safranin was solubilized with 100 μL of a solution containing 80 % ethanol and 20 % acetone (v/v). Ethanol-acetone solutions were transferred to new plates, and the absorbance at 492 nm was measured using a microplate reader (BioRad). Wells containing medium only served as negative controls, and those with cultures without natural compounds served as positive controls. The specific biofilm formation (SBF) was determined according to Niu and Gilbert (2004), using the formula SBF = (AB − CW) / G, in which SBF is the specific biofilm formation, AB is the A 492 of the attached and stained bacteria, CW is the A 492 of the stained control wells containing only bacteria-free medium or with the respective compound (to eliminate unspecific or abiotic A 492 values), and G is the A 600 of cells growth in broth. The SBF values of the treatments were compared with the controls.

Statistical analyses

All experiments were performed in triplicate at least three times. Statistical analyses were performed using SPSS software (version 10.0, SPSS Inc., Chicago, IL). Results were analyzed with an analysis of variance test, and the mean comparison was used for the analyses. Differences between means were considered significant at P values of ≤0.05.

Results and discussion

All compounds exhibited antibacterial activities against four strains of C. jejuni by preliminary antimicrobial assays (data not shown). The MBCs obtained ranged (in μg/mL) from 31 to 125 for EGCG, 150 to 310 for furanone, and 0.3 to 0.35 for the disinfectant (Table 1). The disinfectant exhibited the greatest antibacterial activity, followed by EGCG and furanone.

Table 1 MBC (μg/mL) of three natural compounds against four Campylobacter strains
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The susceptibility of the strains to compounds varied, being C. jejuni 238ip more susceptible than the other strains. This variability in susceptibility to natural antimicrobials has been observed previously (Juntunen et al. 2013 ; Castillo et al. 2011).

Subinhibitory concentrations of the compounds did not affect the growth of C. jejuni nor V. harveyi (data no shown). These results agree with those reported by Lee et al. (2009), in which subinhibitory concentrations of EGCG did not affect the growth of E. coli O157:H7.

Al-2 activity was produced by all strains of C. jejuni tested, at levels near to those produced by V. harveyi BB152 (positive control, Fig. 1). V. harveyi BB170 without CFSNs did not show significant values of luminescence (data no shown). When antimicrobial compounds were added to the C. jejuni supernatants, decreases in bioluminescence were observed. All of the compounds resulted in significant (P ≤ 0.05) decreases in AI-2 activity (bioluminescence) for each strain, ranging from 90 to 96 % for EGCG, 60 to 96 % for furanone, and 95 to 99 % for the disinfectant (Fig. 1). The efficacy of EGCG to decrease bioluminiscence is in accordance with previous studies in which this compound inhibited AI-2 activity by 94 % in E. coli O157:H7 (Lee et al. 2009).

Fig. 1
figure 1

Effect of natural compounds on bioluminescence (AI-2 activity) in supernatants of C. jejuni strains. Supernatants of V. harveyi BB152 served as a positive control

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All of the compounds elicited dose-related reductions in C. jejuni motility (Table 2). Motility of treated cultures was compared to those of nontreated cultures and reduction determined. The greatest reduction in motility for each compound was observed at 75 % of its MBC. The magnitude of reduction varied among the compounds and C. jejuni strains, ranging from 20 to 62 % for EGCG, 19 to 54 % for furanone, and 26 to 57 % for the disinfectant. In general, EGCG was more effective than the disinfectant (Table 2). As expected, the nonmotile C. jejuni 180ip strain did not show motility during the assays at these conditions (data not shown).

Table 2 Reduction (%) of motility of three strains of C. jejuni by natural compounds at subinhibitory concentrations
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All compounds significantly reduced the specific biofilm formation at 75 % of their MBCs (P ≤ 0.05) (Fig. 2). Inhibition of specific biofilm formation was concentration-dependent for each compound. For example, each compound had less effect at 50 % of the MBC than at 75 %. At 25 % of the MBC, no significant inhibition (P ≥ 0.05) of biofilm formation by any compound was observed compared to the controls (data not shown). For all strains, the disinfectant showed the greatest inhibition of biofilm formation. The nonmotile strain C. jejuni 180ip did not form biofilms under the conditions tested. Since subinhibitory concentrations of the compounds did not affect the growth of C. jejuni nor V. harveyi, it could be suggested that diminution in motility, biofilm formation, or bioluminescence is not due to decrease in cell number or cell death.

Fig. 2
figure 2

Biofilm formation of C. jejuni after addition of natural compounds (at 75 % MBC)

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Increasing interest in identifying new strategies to control pathogenic microorganisms and concerns about the emergence of antibiotic-resistant strains has generated much interest in the antimicrobial activity of natural plant products (Girennavar et al. 2008; Daglia 2012). Natural products, such as plant extracts, or isolated compounds provide many new opportunities to control microbial growth (Negi 2012). Combined with advances in chemistry, the increased knowledge of natural products has led to the development of novel preservatives and therapeutics with different mechanisms that specifically target virulence factors (Clatworthy et al. 2007). QS represents a key target for this approach because the interruption of the QS signaling processes can limit or halt the survival of bacteria (Roy et al. 2010).

Many furanones possess antimicrobial properties through their anti-QS activities (Hentzer et al. 2002), inhibit swarming and biofilm formation in E. coli, and provide protection against infections in some marine organisms (Roy et al. 2011). EGCG possesses antiviral, antifungal, and antimicrobial activities (Daglia 2012). It inhibits growth of several bacterial species, such as Vibrio cholerae, Streptococcus mutans, Clostridium perfringens, and E. coli (Daglia 2012; Cui et al. 2012). Citrol K Ultra®, a disinfectant based on citrus extracts, also exhibits antimicrobial activity and has been used to disinfect parsley and cilantro (Orue et al. 2013). In this study, these three compounds inhibited the growth of all of the tested C. jejuni strains, with the disinfectant exhibiting the lowest MBC concentration (0.3 μg/mL).

The MBCs of 2(5H)-furanone against the different bacterial strains ranged from 150 to 310 μg/mL, which are greater than the minimal inhibitory concentrations (MICs) reported for other furanones, such as dimethylhydroxy-furanone against E. coli (40–80 μg/mL), Staphylococcus aureus (20–80 μg/mL), Pseudomonas aeruginosa (20–80 μg/mL), and Enterococcus faecium (40–120 μg/mL) (Sung et al. 2007). Other furanones, such as [2,5-dimethyl-4-hydroxy-3(2H)-furanone and 5-(1,2) di-hydroxyethyl-3,4dihydroxy-2(5H) furanone], have been shown to exhibit greater MICs (800–2,500 μg/mL for inhibiting the growth of C. perfringens strains (Novak and Fratamico 2004). Typically, the MBC is higher than the MIC. Differences in the antimicrobial activities of the various furanones could be due to variations in their molecular structures. Variations in the three alkyl chains of these compounds impact their antimicrobial activity. In particular, compounds with ethyl, butyl, and hexyl side chains reportedly show strong activities (Steenackers et al. 2010).

The 2(5H)-furanone used in this study has a furanone ring structure with no side chains. It has antimicrobial activity against Pseudomonas aeuruginosa, and subinhibitory concentrations have been shown to affect motility (Shobharani and Agrawal 2010). 2-(5H)-furanone produced a 60–90 % decrease in bioluminescence. This finding could indicate a reduction of Al-2 activity, which has been suggested for other furanones in C. perfringens (Novak and Fratamico 2004), Salmonella typhimurium (Steenackers et al. 2010), and P. aeruginosa (Shobharani and Agrawal 2010). This is the first report of the anti-QS activity of 2(5H)-furanone in Campylobacter.

EGCG and the disinfectant showed the greatest inhibition (90–96 %) of AI-2 activity in C. jejuni. EGCG was previously shown to inhibit the AI-2 activity in E. coli O157:H7 by 94 % (Lee et al. 2009). Bacterial motility was also reduced by the compounds analyzed. This finding is consistent with previous work demonstrating that 2(5H)-furanone and bromofuranone reduced motility in P. aeruginosa (Shobharani and Agrawal 2010), whereas halogenated furanones decreased motility in Serratia liquefasciens (Hjelmgaard et al. 2003). A decrease in biofilm formation at different furanone concentrations has been reported in S. typhimurium, particularly furanones with side chains (Steenackers et al. 2010). This variation in biofilm reduction could be attributed to the bromination patterns of the furanone ring structure and/or to the alkyl chain length at the three position on the ring (Hjelmgaard et al. 2003; Steenackers et al. 2010).

Conclusion

In conclusion, the results from this study provide evidence for the bactericidal activity of 2(5H)-furanone and the disinfectant and support the activity of EGCG against C. jejuni. These compounds interfered with QS and other virulence factors, such as motility and biofilm formation, and could be alternatives to help control C. jejuni. Studies are underway to determine (1) if the inhibition of motility and biofilm formation resulted from disturbance of QS and (2) the safety and efficacy of the compounds for food applications and disease treatments.