(-)-α-Pinene reduces quorum sensing and Campylobacter jejuni colonization in broiler chickens.

Campylobacter jejuni is one of the most prevalent causes of bacterial gastroenteritis worldwide, and it is largely associated with consumption of contaminated poultry. Current Campylobacter control measures at the poultry production level remain insufficient, and hence there is the need for alternative control strategies. We evaluated the potential of the monoterpene (-)-α-pinene for control of C. jejuni in poultry. The antibacterial and resistance-modulatory activities of (-)-α-pinene were also determined against 57 C. jejuni strains. In addition, the anti-quorum-sensing activity of (-)-α-pinene against C. jejuni NCTC 11168 was determined for three subinhibitory concentrations (125, 62.5, 31.25 mg/L) over three incubation times using an autoinducer-2 bioassay based on Vibrio harveyi BB170 bioluminescence measurements. The effects of a subinhibitory concentration of (-)-α-pinene (250 mg/L) on survival of C. jejuni, and in combination with enrofloxacin on fluoroquinolone resistance development in C. jejuni, were determined in a broiler chicken model, by addition of (-)-α-pinene to the broiler water supply. The reduction of C. jejuni numbers by (-)-α-pinene was further determined in broiler chickens that were colonized with either fluoroquinolone-susceptible or -resistant strains, by direct gavage treatment. We observed weak in vitro antimicrobial activity for (-)-α-pinene alone (MIC >500 mg/L), but strong potentiating effects on antibiotics erythromycin and ciprofloxacin against different Campylobacter strains (>512 fold change). After 24 h of treatment of C. jejuni with (-)-α-pinene, its quorum-sensing signaling was reduced by >80% compared to the untreated control. When given in the drinking water, (-)-α-pinene did not show any significant inhibitory effects on the level of C. jejuni in the colonized chickens, and did not reduce fluoroquinolone resistance development in combination with enrofloxacin. Conversely, when (-)-α-pinene was administered by direct gavage, it significantly reduced the number of fluoroquinolone susceptible C. jejuni in the colonized broiler chickens. These results demonstrate that (-)-α-pinene modulates quorum-sensing in Campylobacter, potentiates antibiotics against different Campylobacter strains, and reduces Campylobacter colonization in broiler chickens.

Introduction

Campylobacter jejuni represents a food safety hazard worldwide. It can cause campylobacteriosis, which is one of the most widespread bacterial foodborne zoonoses reported for the European Union and the United States [1-3]. Campylobacteriosis is commonly associated with ingestion of contaminated poultry, water, or milk, and manifests as acute watery/bloody diarrhea, fever, and cramps. This can also lead to post-infection development of the severe neurological condition known as Guillain-Barre syndrome [1]. An additional risk is the increasing antimicrobial resistance in Campylobacter. In particular, Campylobacter resistance to fluoroquinolones and macrolides compromises effectiveness of antibiotic therapies and poses a heightened food safety concern in the food chain [2,4].

Currently, there are no fully effective and practical measures for the control of poultry contamination with the avian commensal C. jejuni. Control of Campylobacter includes pre-harvest measures on poultry farms and post-harvest approaches in processing plants. Pre-harvest biosecurity and hygiene measures can be used to prevent entrance of Campylobacter onto a farm and to limit its spread between flocks, whereas post-harvest measures focus on decontamination of carcasses [5-7].

To mitigate transmission of Campylobacter from food animals to humans through the food supply chain, effective pathogen control measures are needed. These must be designed to reduce the Campylobacter load at the farm and/or slaughterhouse level, with emphasis on poultry production, where Campylobacter resides as a commensal [2,4,8]. Even a relatively small reduction in C. jejuni numbers in the chicken cecum by 1 log10 CFU can reduce the public health risk by more than 50% [8].

A number of natural products have been shown to have anti-Campylobacter activities and have been studied as feed additives, such as essential oils and their components [9,10]. The majority of studies have been focused on the bactericidal aspects of the antimicrobial actions of natural compounds, while their potential for reduction of pathogen virulence through inhibition of efflux pumps, quorum sensing or other factors contributing to colonization of a host, remains largely unexplored [11,12].

In C. jejuni, quorum sensing is mediated by the furanosyl borate diester autoinducer-2 (AI-2) signal that is produced as a result of the action of the S-ribosylhomociateinase LuxS, encoded by the luxS gene [13]. The C. jejuni mutant lacking the luxS gene shows impaired biofilm formation, motility, resistance against oxidative stress, invasion of Caco-2 cells, virulence in the host, and colonization of the chicken intestine [13-18]. This suggests that inhibition of C. jejuni quorum sensing in the host might result in reduction of C. jejuni in the feces, and thus control C. jejuni spread in the environment.

Only a few plant extracts have been reported to show anti-quorum-sensing effects in C. jejuni (e.g., citrus extracts, Evodia ruticarpa extracts) to date [19,20].

In a previous study, we also demonstrated efflux-inhibitory and resistance-modulatory activities of the monoterpene (-)-α-pinene in Campylobacter [21]. These findings suggest that plant extracts, such as (-)-α-pinene, modulate multiple physiological functions in C. jejuni. However, the effects of these plant extracts have not been examined using an in vivo system, which would allow for determination of their potential use in food-animal production. In this study we further investigated (-)-α-pinene bioactivities, including: (i) inhibition of C. jejuni quorum sensing in vitro; (ii) modulation of C. jejuni resistance to fluoroquinolones in broiler chickens; and (iii) reduction of C. jejuni colonization in broiler chickens.

Materials and methods

Bacterial strains and growth conditions

The Campylobacter jejuni strains shown in S1 and S2 Tables were isolated and characterized by Luangtongkum et al. [22], and were stored at -80°C in 80% Mueller Hinton broth (MHB: Oxoid, UK) with 20% glycerol. They were then grown on Mueller-Hinton agar (MHA; Oxoid, UK) at 42°C under microaerobic conditions (5% O2, 10% CO2, 85% N2) for 24 h. The second passage from each culture was used in the experiments. When necessary, MHA was supplemented with selective medium (SR01176; Oxoid, UK) and growth medium (SR0232E; Oxoid, UK) (MHA-SS), 30 mg/L kanamycin (Merck, Germany), or 4 mg/L ciprofloxacin (Merck, Germany). The Vibrio harveyi BB170 reporter strain [19,23] was grown on autoinducer bioassay (AB) medium at 30°C, which contained 17 g/L NaCl (Merck, Germany), 12.3 g/L MgSO4 (Merck, Germany), 2 g/L casamino acids (BD Bacto; Fisher Scientific), 1 mM K2HPO4 (Kemika, Croatia), 0.1 mM L-arginine (Sigma Aldrich, Germany), and 1% (v/v) glycerol (Kemika, Croatia).

Antimicrobial and resistance-modulatory activities of (-)-α-pinene in vitro

The minimal inhibitory concentrations (MICs) of (-)-α-pinene (Sigma Aldrich, Germany) were determined against all of the 57 C. jejuni strains that were sourced according to S1 Table, using the broth microdilution method, as described previously [21]. The reported MIC50 and MIC90 values represent the MICs that inhibited at least 50% and 90%, respectively, of the tested strains. The resistance-modulatory activity of (-)-α-pinene was determined in combination with the clinically relevant antibiotics ciprofloxacin and erythromycin (Fluka Chemie, Germany), using the broth microdilution method [21]. (-)-α-Pinene was added to these antibiotics at the subinhibitory concentration of 125 mg/L. The MICs were determined, along with the fold-changes (FC) between the MICs of the antibiotics alone and their MICs with the addition of (-)-α-pinene. These were calculated according to Eq (1): where MICAb is the MIC of the antibiotic alone, and MICAbAp is the MIC of the antibiotic in the presence of 125 mg/L (-)-α-pinene. FC ≥2 was considered as indicative of biologically significant resistance modulation.

Quorum-sensing inhibition in vitro

To determine the influence of (-)-α-pinene on C. jejuni quorum sensing, autoinducer-2 bioassays were performed. C. jejuni NCTC 11168 and C. jejuni 11168ΔluxS (negative control; [18]) cultures in MHB were adjusted to OD600 0.1. The (-)-α-pinene stock solutions were prepared in 100% dimethylsulfoxide (DMSO) at 6.25 g/L, 12 g/L, and 25 g/L. Fifty microliters of each stock was added to 10 mL of each culture for the final (-)-α-pinene concentrations of 31.25 mg/L, 62.5 mg/L, and 125 mg/L. Untreated cultures were used as controls. The cultures were incubated under microaerobic conditions at 42°C for 24 h. Samples of 3 mL were taken after 4 h, 8 h, and 24 h, and filter sterilized using 0.2-μm syringe filters (Sartorius, Germany), for the cell-free supernatants.

The autoinducer-2 bioassay was performed as previously described [19], with some modifications. The quorum-sensing inhibition bioassays were carried out using a V. harveyi BB170 reporter strain [23]that was grown for 16 h at 30°C and 150 rpm, and used at the final concentration of 5 ×104 CFU/mL in AB medium. Filter sterilized C. jejuni cell-free supernatants were added to the suspensions of the reporter strain to a final concentration of 10% (v/v) (i.e., 20 μL cell-free supernatant added to 180 μL reporter strain suspension). Sterile medium was used as the blank (10% [v/v] MHB, 90% [v/v] AB medium). Kinetic measurements were carried out for the bioluminescence signals of V. harveyi BB170 produced as a result of the presence of the quorum-sensing signal that originated from the C. jejuni cell-free supernatants. The relative luminescence signals were measured at 15-min intervals over 20 h at 30°C, in white microtiter plates (Nunc, Thermo Scientific) incubated in a microplate reader (Varioskan Lux; Thermo Scientific).

Vibrio harveyi produces a background luminescence signal that increases with the concentration of the culture. To define the most stable point of signal production, V. harveyi growth and signal production was measured in AB supplemented with MHB (180 μL:20 μL) at 30°C. The signal stabilized when V. harveyi entered the stationary phase (S1 Fig). The time point when V. harveyi enters the stationary phase (after 9 h incubation) was used in the calculation of the quorum-sensing signals attributed to C. jejuni.

The relative luminescence signals were interpreted as the quorum-sensing signal in the C. jejuni cell-free supernatants (i.e., a higher signal indicated a higher concentration of quorum-sensing signaling molecules produced by C. jejuni), and are shown in S2 Fig.

Cell-free supernatants from C. jejuni 11168ΔluxS, a mutant that cannot produce the quorum-sensing signal (AI-2), were used as the negative control, and fresh MHB as the blank. To determine the inhibition rates of the quorum sensing by (-)-α-pinene, the blank values were subtracted from all of the test sample values. These corrected test values were used to calculate the reduction in quorum sensing using Eq (2): The experiments were performed as three independent biological replicates and three technical replicates.

Broiler chicken colonization with C. jejuni

Broiler chicks (Cornish Rock strain, unspecified sex) were obtained from the Welp Hatchery in Iowa (USA) on the day of hatching, and were divided into four groups of 10 broilers each. The broiler chickens were kept in sanitized wire-floored cages (each group, n = 10/cage), and provided with feed and water ad libitum. Cloacal swabs were taken from each broiler chicken prior to the experiment and plated onto MHA-SS to confirm that they showed no Campylobacter colonization prior to inoculation. No Campylobacter was detected in any of the broiler chickens tested. At the age of day 5, each bird was inoculated with 3.6 ×106 CFU C. jejuni NCTC 11168 by oral gavage. To confirm colonization, cloacal swabs were collected 3 days after the inoculation.

At the age of day 8, medicated water was given to birds for 5 consecutive days to evaluate the synergistic effects of (-)-α-pinene and enrofloxacin on Campylobacter fluoroquinolone resistance development. Since enrofloxacin and (-)-α-pinene were dissolved in DMSO, the medicated water contained 0.5% DMSO for all groups, with the following additions for each group: (1) none (DMSO; control group); (2) 250 mg/L (-)-α-pinene (AP); (3) 50 mg/L enrofloxacin (ENRO) (Sigma Aldrich); and (4) 250 mg/L (-)-α-pinene and 50 mg/L enrofloxacin (ENRO+AP). Cloacal swabs were collected every other day, and 3 days after (day 16 of age) the final day of the treatment for Campylobacter culture.

As the culture results of the cloacal swabs showed, all of the birds in all of the groups were colonized by C. jejuni. In addition, enrofloxacin treatment resulted in development of fluoroquinolone resistance in C. jejuni in the treated groups (FQ-R; groups 3 (ENRO) and 4 (ENRO+AP) above), while the groups that were not treated with enrofloxacin remained colonized by fluoroquinolone sensitive C. jejuni (FQ-S; groups 1 (DMSO) and 2 (AP) above). To further determine the effects of (-)-α-pinene on susceptible and resistant C. jejuni in vivo, one group of each category (groups 2 and 4) were given an additional 250 mg/L (-)-α-pinene directly by oral gavage (i.e., the FQ-S treated and FQ-R treated groups) while the other two (groups 1 and 3) did not receive any (-)-α-pinene (i.e., the FQ-S untreated and FQ-R untreated groups). The gavage water (0.4 mL/bird/day) was started at the age of 18 days for 3 consecutive days, and it contained 0.5% DMSO for all four groups. Direct gavage treatment was used to minimize the variability of the dosing between the broiler chickens. All of the broiler chickens were sacrificed at 21 days of age, at which time cecum contents were collected for Campylobacter culture.

For determination of Campylobacter numbers, all of the fecal swabs and the cecum contents collected were suspended in MHB (1 mL MHB/swab with 100 mg feces), serially diluted, plated onto MHA-SS (for total C. jejuni numbers) and onto MHA-SS supplemented with 4 mg/L ciprofloxacin (for fluoroquinolone-resistant C. jejuni), and incubated at 42°C under microaerobic conditions for 48 h. The detection limit of the culture method for C. jejuni was 100 CFU/g feces. To further confirm the emergence of fluoroquinolone-resistant C. jejuni mutants, colonies from MHA-SS were also collected for each group at every sampling, and antimicrobial sensitivity testing was performed using E-test strips (0.002–32 mg/L ciprofloxacin; AB Biodisk, Sweden).

Ethics statement

All of the animal protocols and procedures used in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Iowa State University (Ames, Iowa, USA) before the start of the experiments. The approved protocol identification number is: 2-07-6304-G. The animal care and use protocol used in this study adhered to regulations and guidelines provided in the “Guide for the Care and Use of Laboratory Animals”, 8th edition, and the “Guide for the Care and Use of Agricultural Animals in Research and Teaching”, 3rd edition.

Statistical analyses

All of the data were tested for normality with Kolmogorov-Smirnov and Shapiro-Wilk tests. The statistical significances of the quorum-sensing inhibition and antimicrobial and resistance-modulatory activities were calculated using one-way ANOVA with Tukey’s post-hoc tests. The associations between antibiotic resistance and resistance modulation were calculated using Chi-squared tests with Cramer’s V strength tests. Differences in colonization between the treated and untreated broiler chickens were analyzed using Student’s t-tests. Emergence of fluoroquinolone-resistant mutants in groups was compared using Student’s t-tests. All of the analyses were performed using the SPSS software, version 21 (IBM Corp., Armonk, NY, USA).

Results

(-)-α-Pinene shows weak antimicrobial activity but strong resistance-modulatory activity against C. jejuni in vitro

To evaluate the clinical relevance of previously reported antimicrobial and resistance-modulatory activities of (-)-α-pinene [21], these activities were tested across 57 broiler, turkey, and human C. jejuni strains, in addition to the reference strain (NCTC 11168), which were sourced as listed in S1 Table. The following criteria were defined for the antimicrobial activities of (-)-α-pinene alone: high: MIC ≤31.25 mg/L; intermediate: MIC from 62.5 mg/L to 1000 mg/L; low: MIC at 2000 mg/L; none: MIC >2000 mg/L. Based on these criteria, and collectively considering these 57 C. jejuni strains, (-)-α-pinene alone showed low antimicrobial activity, with the overall MIC50 of 2000 mg/L (concentration of (-)-α-pinene that inhibited at least 50% of the strains; Table 1). Considering the strains individually, the majority of these strains showed low antimicrobial activities of (-)-α-pinene (n = 39; 68%), with no effects seen against 12% (n = 7) (Table 1). These data thus demonstrate the relatively weak antimicrobial activity of (-)-α-pinene alone against C. jejuni.

Table 1

Antimicrobial and resistance modulatory activity of (-)-α-pinene with antibiotics ciprofloxacin and erythromycin in 57 Campylobacter jejuni strains from chicken meat (strain code, CB), turkey meat (strain code, CT), human feces (strain codes F, X) and the reference strain NCTC 11168.

Strain	(-)-α-Pinene	Ciprofloxacin			Erythromycin
code	MIC (mg/L)	MIC (mg/L)		Fold	MIC (mg/L)		Fold
	Alone	Alone	Plus (-)-α-pinenea	changeb	Alone	Plus (-)-α-pinenea	changeb
CB1:6	1000	16	<0.125	>128	0.5	<0.002	>256
CB1:14	1000	16	<0.125	>128	0.5	<0.002	>256
CB1:18	1000	16	<0.125	>128	0.5	0.125	4
CB2:6	2000	64	16	4	0.5	0.06	8
CB2:8	2000	64	32	2	0.5	0.25	2
CB2:11	1000	8	4	2	0.5	0.25	2
CB3:1	2000	0.125	0.06	2	0.25	0.06	4
CB3:5	2000	0.25	0.06	4	0.5	0.25	2
CB 4:21	1000	0.06	0.03	2	0.06	<0.002	>32
CB 4:22	2000	0.125	0.001	128	0.06	0.002	32
CB 6:8	2000	0.06	0.008	8	0.125	<0.002	>64
CB 6:9	2000	0.06	0.03	2	0.125	0.06	2
CB 6:26	2000	0.06	0.03	2	0.25	0.125	2
CB 7:15	1000	8	<0.06	>128	0.25	<0.002	>128
CB 7:21	2000	8	1	8	0.125	<0.002	>64
CB 8:14	2000	0.06	0.001	64	0.125	0.002	64
CB 8:15	1000	0.06	0.001	64	0.5	<0.002	>256
CT 1:1	2000	16	1	16	0.5	0.03	16
CT 1:9	1000	16	2	8	0.5	0.06	8
CT 2:2	2000	16	<0.06	>256	256	<1	>256
CT 3:5	2000	0.06	<0.001	>64	0.03	<0.002	16
CT3:11	500	4	<0.06	>64	-	-	-
CT3:19	2000	16	<0.06	>256	512	<1	>512
CT4:4	2000	16	8	2	128	64	2
CT4:14	2000	8	8	1	128	64	2
CT5:2	2000	16	4	4	256	256	1
CT5:8	2000	8	2	4	256	16	16
CT5:10	2000	16	<0.06	>256	256	<1	>256
CT5:12	2000	16	4	4	256	32	8
CT5:18	2000	16	4	4	256	64	4
CT 6:18	2000	8	0.5	16	128	<1	>128
CT 6:8	2000	16	2	8	256	<1	>256
CT 6:16	2000	0.03	<0.001	>32	128	<1	>128
CT 7:2	2000	0.03	<0.001	>32	0.06	0.002	32
CT 8: 28	2000	8	1	8	0.25	<0.008	>32
CT 8:29	>2000	4	<0.06	>64	64	8	8
CT 8:22	2000	4	0.5	8	0.5	0.5	1
CT 9:14	2000	0.25	<0.002	>128	2	<0.008	>256
CT 10:18	1000	0.06	<0.002	>32	0.25	<0.008	>32
CT 9:21	1000	0.125	<0.002	>64	2	<0.008	>256
F6501	>2000	0.125	0.06	2	0.25	0.125	2
H2958	2000	0.125	0.03	4	0.5	0.25	2
M63885	2000	0.25	<0.002	>128	0.5	<0.008	>64
T59822	2000	0.125	0.016	8	0.25	0.06	4
W14861	>2000	0.25	0.25	1	2	2	1
X60179	2000	8	<0.06	>128	0.25	<0.002	>128
F15871	>2000	0.125	0.06	2	1	0.5	2
W11805	2000	0.06	0.031	2	0.25	0.06	4
M402	>2000	0.06	0.031	2	1	0.5	2
W28752	2000	0.06	0.008	8	0.5	<0.002	>256
M33323	2000	0.06	0.06	1	0.25	0.25	1
W64861	2000	0.125	0.06	2	0.25	0.25	1
M76297	2000	0.06	0.03	2	0.25	0.125	2
E46972	>2000	1	<0.002	>512	0.25	<0.008	>32
M36292	2000	0.06	<0.002	>32	0.25	<0.008	>32
X7199	>2000	16	16	1	0.25	0.125	>32
NCTC 11168	2000	0.06	0.03	2	0.25	0.06	4
MIC90c	2000	16	4	4	256	16	16
MIC50d	2000	0.25	0.06	4	0.5	0.06	8

a Addition of (subinhibitory) 125 mg/L (-)-α-pinene

b Fold change (improvement) of MIC with addition of 125 mg/L (-)-α-pinene

c Concentration that inhibits 90% of the tested strains [24]

d Concentration that inhibits 50% of the tested strains [24]

The resistance-modulatory activity of (-)-α-pinene in C. jejuni with two clinically important antibiotics (i.e., ciprofloxacin, erythromycin) was tested at the subinhibitory concentration of 125 mg/L (-)-α-pinene. These data are reported as fold-changes (FC) in terms of the decrease in the MICs of the antibiotics when combined with (-)-α-pinene (Table 1). The following criteria were set for the resistance-modulatory activities in terms of the fold-changes: high: ≥32; intermediate: <32 to ≥8; low: <8 to ≥2; and no activity, 1.

The FC differed among the strains, from 1 (i.e., no activity) to >512 (i.e., high activity). When combined with ciprofloxacin, (-)-α-pinene showed strong and intermediate resistance-modulatory activities in 39% (n = 22) and 18% (n = 10) of the strains, respectively. The susceptibility to ciprofloxacin was affected marginally by (-)-α-pinene (i.e., low activity) in 37% (n = 21) of the strains, and not affected at all in 7% (n = 4) of the strains. The antimicrobial activity of erythromycin was enhanced by (-)-α-pinene in the majority of the tested strains. In 46% (n = 26), (-)-α-pinene showed high resistance-modulatory activity; in 13% (n = 7), intermediate, and in 32% (n = 18), low activity. (-)-α-Pinene did not increase the susceptibility to erythromycin in 9% (n = 5) of the tested strains. It was interesting to note that only strains M33323 and W14861 did not show any changes in susceptibility to both ciprofloxacin and erythromycin when combined with (-)-α-pinene.

We then compared the resistance-modulatory activity of (-)-α-pinene for ciprofloxacin and erythromycin with the available antibiotic susceptibility data for a range of antibiotics (i.e., ampicillin, tetracycline, kanamycin, gentamicin, erythromycin, clindamycin, ciprofloxacin, nalidixic acid, norfloxacin), using 37 of the broiler and turkey strains (S2 Table). Here, no significant associations were seen between the susceptibilities to any specific antibiotic and the resistance-modulatory activities of (-)-α-pinene. Thus, these resistance-modulating activities of (-)-α-pinene did not depend on the susceptibility to any of the antibiotics tested.

Campylobacter jejuni quorum sensing is inhibited by (-)-α-pinene in vitro

To determine the potential of (-)-α-pinene for inhibition of quorum sensing, C. jejuni NCTC 11168 was treated with three subinhibitory concentrations of (-)-α-pinene (i.e., 31.25, 62.5, 125 mg/L) for 4 h, 8 h, and 24 h. The reductions in the quorum-sensing signaling molecules produced in the treated cultures were calculated and compared to that for the untreated cultures. Inhibition of C. jejuni quorum sensing was seen for all of these samples treated with (-)-α-pinene, regardless of the concentration added, and at all time-points (Fig 1). After 8 h treatment with (-)-α-pinene, the quorum-sensing inhibition was in the same range for all of the (-)-α-pinene concentrations used (10%-13% inhibition; p >0.05). After 4 h and 8 h of treatments, the highest quorum-sensing inhibition by (-)-α-pinene did not exceed 20%. After 24 h of treatment, there was higher quorum-sensing inhibition in all of the samples treated with (-)-α-pinene, compared to the shorter incubation times (p <0.01). The 24-h treatment with 31.25 mg/L (-)-α-pinene resulted in 36% inhibition of quorum sensing, while the higher treatments with 62.5 mg/L and 125 mg/L (-)-α-pinene showed 83% (p = 0.001) and 85% (p <0.001) inhibition, respectively, of quorum sensing compared to the untreated control. These data thus showed concentration-dependent quorum-sensing inhibitory activities of (-)-α-pinene, which was emphasized by the prolonged treatment times.

Fig 1

Time-course of (-)-α-pinene inhibition of quorum sensing in C. jejuni NCTC11168.

Data are means ±standard deviation of relative reduction of quorum sensing signal (as luminescence of V. harveyi BB170) in the treated C. jejuni cell free supernatants (CFS) versus the untreated C. jejuni CFS, calculated from three replicates. * p <0.05, *** p ≤0.001.

(-)-α-Pinene does not reduce fluoroquinolone resistance development when added to enrofloxacin in broiler chickens

Although the use of enrofloxacin is prohibited in poultry production in the USA due to its rapid induction of fluoroquinolone resistance in C. jejuni [25], it is still used in veterinary medicine in the European Union [26]. Based on the obvious activity of (-)-α-pinene in modulating fluoroquinolone resistance (Table 1), we investigated whether a subinhibitory concentration of (-)-α-pinene can delay development of fluoroquinolone resistance of C. jejuni in the chicken model, with the addition of (-)-α-pinene and enrofloxacin into the broilers water supply. Here, the (-)-α-pinene concentration used was doubled (but still subinhibitory) compared to the in vitro studies, to ensure sufficient concentration in the intestinal tract in chickens.

As treatments with enrofloxacin are known to induce fluoroquinolone resistance in Campylobacter [25], these analyses included enumeration of both the total numbers of Campylobacter and the proportion of ciprofloxacin-resistant Campylobacter in each broiler chicken fecal sample. The results showed that all of the Campylobacter isolates (100%), in all of the colonized birds treated with enrofloxacin developed fluoroquinolone resistance at all of the sampling times after the treatment had begun, regardless of inclusion of (-)-α-pinene in the drinking water. In contrast, Campylobacter isolates from the broiler chicken groups treated with DMSO and (-)-α-pinene alone (i.e., no enrofloxacin treatment) did not develop any resistance (0%) to fluoroquinolones at any point during the experiment. During these treatments, the mean Campylobacter numbers for (-)-α-pinene alone (4.37 log10 CFU/g) tended to be lower than that of the DMSO control (5.05 log10 CFU/g); however, there was wide variability within each of these treatment groups, so these data did not reach statistical significance (Fig 2).

Fig 2

Time-courses of the effects of (-)-α-pinene in the water supply of broiler chickens inoculated with C. jejuni NCTC 11168 3 days before (-)-α-pinene treatment (started day 0).

Data are C. jejuni counts (log10 CFU/g feces) in cloacal swabs from individual broiler chickens in the treatment groups: DMSO, no treatment control; AP, 250 mg/L (-)-α-pinene; ENRO, 50 mg/L enrofloxacin; ENRO+AP, combination of 50 mg/L enrofloxacin and 250 mg/L (-)-α-pinene. The detection limit of the culture method was approximately 2 log10 CFU/g feces, and the means are indicated by the horizontal lines.

These data showed that (-)-α-pinene did not have any resistance-modulatory activity in vivo, nor did it reduce the rapid development of fluoroquinolone resistance of C. jejuni after exposure to enrofloxacin. However, addition of (-)-α-pinene alone to the broiler chicken water supply reduced the average numbers of Campylobacter in the already colonized broiler chickens, although this did not reach statistical significance. Of note, with the (-)-α-pinene here added to the water supply, the amount of (-)-α-pinene ingested by each broiler chicken could not be controlled, which is likely to explain the large variations in these data.

Reduction of Campylobacter in broiler chicken cecum after direct gavage with (-)-α-pinene

To better evaluate whether (-)-α-pinene can modulate C. jejuni colonization in the same broiler chicken experiment described above, chickens colonized by FQ-S Campylobacter and FQ-R Campylobacter were treated with (-)-α-pinene by direct oral gavage. With this treatment, the amount of (-)-α-pinene consumed by each animal was better controlled.

These data indicated that there were significantly lower C. jejuni counts in the broiler chickens colonized with FQ-S C. jejuni when treated with (-)-α-pinene (FQ-S treated), with a reduction of 0.8 log10 CFU/g unit (p = 0.028; Fig 3) compared to the untreated group (FQ-S untreated). No significant differences were seen between the nontreated and (-)-α-pinene–treated groups that were colonized with FQ-R C. jejuni (FQ-R untreated, FQ-R treated), although a slight mean reduction from 7.0 to 6.6 log10 CFU/g cecum content (p = 0.095; Fig 3) was observed in the treated group compared to untreated. These data show that (-)-α-pinene reduced colonization of the FQ-S C. jejuni in broiler chickens when administered by direct gavage, but it had no significant effect on FQ-R C. jejuni.

Fig 3

Campylobacter counts (log10 CFU/g) in the cecum content of broiler chickens following treatments without and with a subinhibitory (-)-α-pinene concentration via direct gavage for 3 consecutive days (0.4 mL, 250 mg/L, daily).

The samples were collected 24 h after the last treatment, and cultured for Campylobacter. Data are means ±standard deviation from individual broiler chickens that were colonized with either FQ-S or FQ-R C. jejuni prior to the treatment. FQ-S/FQ-R untreated, fluoroquinolone-sensitive/resistant C. jejuni (controls); FQ-S/FQ-R treated, fluoroquinolone-sensitive/resistant C. jejuni plus treatment with (-)-α-pinene. *p <0.05.

Discussion

The effects of (-)-α-pinene have been seen to be versatile, from antioxidative to cell protective [27], and to anti-cancer [28], with only weak antimicrobial activities reported previously [29]. In the present study, we showed that the concentrations of (-)-α-pinene needed for antimicrobial effects against C. jejuni were high, and were similar for all of the strains (n = 57) tested regardless of host origin (e.g., chicken, turkey, human) and susceptibility profiles to a range of antibiotics. These data confirm the observations of Kovač et al. [21], where (-)-α-pinene also showed weak antimicrobial activity against nine C. jejuni strains tested. We also confirmed earlier indications of the strong in-vitro resistance-modulatory activity of (-)-α-pinene for clinically important antibiotics (i.e., ciprofloxacin and erythromycin) against this large and diverse collection of C. jejuni strains [21]. These data suggest that (-)-α-pinene may have potential as an adjunctive therapy, in various hosts, to increase the efficacy of macrolides and fluoroquinolones against C. jejuni resistant to these antimicrobials.

Subinhibitory concentrations of (-)-α-pinene have been shown to evoke diverse transcriptional responses in C. jejuni, although the main mechanisms of its resistance-modulatory activity appear to be inhibition of the CmeABC efflux pump and induction of membrane damage [21]. Similarly, Oh and Jeon [30] reported that different monoterpenes can show synergistic effects when combined with ciprofloxacin or erythromycin, due to the modulation of antibiotic influx and efflux in C. jejuni.

Correct functioning of the efflux pumps, such as the CmeABC multidrug efflux pump (the major efflux pump in C. jejuni), is needed not only to enhance bacterial resistance to antibiotics, but to also increase bacterial resistance to bile salts, and thus to facilitate the colonization of the gastrointestinal tract in animals and humans by C. jejuni [31]. This suggests that after exposure to an efflux pump inhibitor (e.g., pinene), C. jejuni sensitivity to antimicrobials can increase, its virulence can decrease, and its colonization can become impaired [32]. We therefore tested the potential of (-)-α-pinene to modulate C. jejuni resistance to fluoroquinolones, to attenuate the development of resistance to fluoroquinolones and impair C. jejuni colonization in vivo in a broiler chicken model.

In the broiler chicken model, (-)-α-pinene did not act as a modulator of C. jejuni resistance when administered together with enrofloxacin, nor did it change the development of resistance in C. jejuni to fluoroquinolones when NCTC 11168 was used as the model strain (Fig 2). Due to the ever-growing antibiotic resistance of C. jejuni [33], the concept of a natural compound that can hinder resistance development or have a synergistic activity with antibiotics would open new and attractive opportunities for combating antibiotic resistance. However, the reality is often more complex, as compounds that demonstrate activity in vitro do not always maintain the same activity in vivo, as additional factors, that cannot be controlled, are introduced.

It has been suggested that a reduction of Campylobacter in the chicken intestine by 1 log10 CFU can reduce the public health risk by 50% to 90%, and a 2 log10 CFU reduction can reduce the risk by >90% [8,34,35]. This can be achieved with natural compounds [10]. Supplementation of poultry feed and water with natural compounds has been shown to reduce Campylobacters in poultry, and in some cases, this has improved animal health and yield as well. For example, feed supplementation with carvacrol and thymol at inhibitory concentrations has shown significant reduction of Campylobacter and Salmonella colonization [36] and growth enhancement in broiler chickens [37]. Grilli et al. [9] lowered Campylobacter counts in the broiler chicken cecum by 1 log10 CFU/g with feed additives of essential oils at 5000 mg/L, which represented an antimicrobial concentration. In the present study, a 0.8 log10 CFU reduction in Campylobacter counts in the broiler chicken cecum was obtained using a lower, and subinhibitory, concentration of (-)-α-pinene (250 mg/L) (Fig 3). This suggests that even lower concentrations of natural compounds, where bioactivity is still observed, can contribute to Campylobacter control in broilers, thus reducing the amount of treatment needed. The C. jejuni colonization reduction by (-)-α-pinene in a subinhibitory concentration can be explained by its efflux pump inhibitory [21] and quorum sensing inhibitory (Fig 1) activities exhibited at sub-inhibitory concentrations in vitro. In C. jejuni, both intact efflux pump activity [31] and quorum sensing [17] are important for colonization of the host, thus can the inhibition of these systems, by an external source such as (-)-α-pinene, contribute to C. jejuni control.

When a substance is introduced into an animal host to promote the reduction of pathogens, it is important to consider both the host response [38] and the response of the pathogen in question to the substance. An important factor for C. jejuni host colonization is cell-to-cell communication, or quorum sensing [13,39]. Disruption of the quorum-sensing system of C. jejuni interferes with its motility and autoagglutination, its production of cytolethal distending toxin, and its host colonization [17,18].

Essential oils and their constituents, such as cinnamaldehyde and cinnamon bark essential oil, can inhibit quorum sensing [40]. Furthermore, in C. jejuni, quorum-sensing inhibitors such as epigallocatechin gallate and extracts of Euodia ruticarpa can reduce motility and biofilm formation [19,20], although the potential in-vivo effects of these on C. jejuni colonization are not known. Brackman et al. [40] demonstrated 65% inhibition of quorum sensing by cinnamaldehyde in Vibrio spp. This was lower compared to that of (-)-α-pinene against C. jejuni in the present study, where the inhibition was >80% when treated with subinhibitory (-)-α-pinene (Fig 1). The quorum-sensing inhibition in Vibrio spp. resulted in down-regulation of virulence factors and weaker cytotoxicity toward Caenorhabditis elegans [40], which indicated that cinnamaldehyde can modulate the pathogen–host interactions. In the present study, we observed changes in pathogen–host interactions in terms of C. jejuni colonization in broiler chicken cecum content after treatment with (-)-α-pinene by direct gavage (Fig 3).

Although the anti-Campylobacter activity of (-)-α-pinene under in vitro conditions was similar against both ciprofloxacin-susceptible and -resistant C. jejuni strains (Table 1), the reduction of FQ-R Campylobacters in the broiler chickens treated with (-)-α-pinene did not reach significance. These findings can be explained by the observations of Luo et al. [25], who showed that FQ-R C. jejuni strains have better fitness in vivo compared to FQ-S strains, and are thus more tolerant to stressors, such as (-)-α-pinene treatment.

The present study stresses the importance of improving the control of Campylobacter in poultry production so as to reduce the public health risk. It also exposes the problem of development of antibiotic resistance in poultry production and the difficulties in the management of human foodborne infections by antibiotic-resistant C. jejuni. Further investigations into the mechanisms of action of natural compounds that might be used for manipulation of pathogen–host interactions and reduction of host colonization in vivo is highly warranted. Furthermore, it is important to consider the effects and mechanisms of action of such compounds at subinhibitory concentrations. This can allow improved prediction of their activity in live systems (i.e., in the animal), where it can be difficult to control the exact amounts that are ingested. For example, for Pseudomonas aeruginosa, inhibitors of quorum sensing have been shown to mitigate infections even without showing strong antibacterial effects [41,42]. This makes the inhibitory effects on quorum sensing an important aspect when searching for new anti-infectious compounds.

For (-)-α-pinene, in C. jejuni it was shown previously to evoke stress and heat-shock responses, to inhibit the multidrug efflux pumps, and to increase membrane permeability [21]. The present study further indicates that it can inhibit quorum sensing in C. jejuni. It is likely that all of these in vitro activities of (-)-α-pinene might have contributed to its in vivo effects observed in the current study on colonization by fluoroquinolone-susceptible C. jejuni in chickens.

Conclusions

The findings from this study indicate that despite showing poor antimicrobial activity against Campylobacter, even at high concentrations, (-)-α-pinene can modulate C. jejuni quorum sensing and colonization of broiler hosts when administered at subinhibitory concentrations. Further in vivo studies are warranted to better evaluate the effects of (-)-α-pinene on colonization by Campylobacter, including different species and strains with different antimicrobial resistance profiles (e.g., erythromycin resistance), as well as various treatment regimens (e.g., therapeutic vs. preventive).

Luminescence production (relative luminescent units, RLU; open squares) and growth of V. harveyi BB170 (OD600nm; filled circles) in AB medium supplemented with 20% MHB.

Data are means ±standard deviation. Framed time points (i.e. 9 h) are those best suited for further evaluation of quorum sensing inhibition.

(DOCX)

Click here for additional data file.

Luminescence of V. harveyi BB170 produced after addition of cell-free supernatants of C. jejuni wild type (wt control; filled symbols) and the ΔluxS mutant (luxS; open symbols) without treatment (circles) and after treatment with (-)-α-pinene at 31.25 mg/L (squares), 62.5 mg/L (triangles), and 125 mg/L (diamonds).

Data are means ±standard deviation for luminescent signal (relative luminescent units, RLU). Framed time point is that used in the calculation of quorum sensing inhibition.

(DOCX)

Click here for additional data file.

Campylobacter jejuni strains used in this study.

(DOCX)

Click here for additional data file.

Susceptibility of Campylobacter jejuni broiler and turkey strains to the range of tested antibiotics, as MICs and corresponding sensitivity (S) or resistance (R).

(DOCX)

Click here for additional data file.

16 Oct 2019

PONE-D-19-20048

(-)-α-Pinene reduces quorum sensing and Campylobacter jejuni colonization in broiler chickens

PLOS ONE

Dear Dr. Smole Možina,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As is often the case, the two reviewers have highlighted different points in their reviews.  I would urge the authors to read these comments carefully for any revision they make, as there are quite a few of them.  However, in terms of what needs to be addressed, the following four points can be made:

1)    Methodological issues - sampling as swabs, but reporting data as if it were faeces, and concerns over the impact of outlier data in the conclusions drawn

2)    Repetition of methods and results - both reviewers comment on this

3)    Justification for issues around the study design - relevance of the study to humans in terms of drug use in humans and chickens

4)    Discussion - some clarification over aspects in regards to framing the results in the current literature

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Reviewer #2: Yes

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Reviewer #1: This paper presents valuable and to a large extent confirmatory findings on the effect of the essential oil component (-)-α-pinene against the most important foodborne pathogen Campylobacter jejuni. The effect of this component on the pathogen was demonstrated on different levels: in vitro through antimicrobial activity, antibiotic resistance modulation, and quorum sensing inhibition; in vivo through a chicken infection model.

The paper is largely based on a previous study of the same research groups (Kovac et al. 2015) in which both the antimicrobial activity and the resistance modulatory activities of (-)-α-pinene in C. jejuni against two fluoroquinolones was investigated. In the present paper, the C. jejuni strain collection was extended to confirm these activities, but surprisingly the strain (NCTC 11168) used for inoculation of the chickens was not included. In addition, now also the inhibition of quorum sensing was shown in a Vibrio harveyi model. Based on these observations, a broiler chicken infection model was used to see if (-)-α-pinene added in the drinking water would reduce the C. jejuni load in the gut. In contract to the in vitro results, no resistance modulatory effect was seen in the chickens, but where are the in vitro data which proof this effect for the inoculation strain ? No effect, either (-)-α-pinene alone or in combination with enrofloxacin, was seen on the colonization level. Only through a direct gavage of (-)-α-pinene, a statistically significant but very modest reduction (< 1 log cfu/g feces) was observed for the ciprofloxacin sensitive C. jejuni population only. In fact, for half of the chickens infected with this sensitive C. jejuni population, the C. jejuni excretion level was comparable with the untreated control. The statistical significance was probably only obtained through one bird showing a considerable lower C. jejuni level. I therefore doubt about the soundness of the statistical analysis using a t-test and the inclusion of the outlier bird.

Although the different categories of results are valuable on their own, there is a general lack of coherence between the results. The quorum sensing results have almost no connection with the other types of data and as they are only observed in vitro, there is no indication that this activity will also appear in vivo and therefore would help to find the mechanism of the observed C. jejuni colonization decrease with (-)-α-pinene direct gavage.

The rationale to look for a synergy between (-)-α-pinene and enrofloxacin in broiler chickens is not clear. Enrofloxacin is used in human medicine, but is not used in broilers to prevent or combat a C. jejuni infection. The observation of the enrofloxacin/ciprofloxacin resistance modulation by (-)-α-pinene in vitro is interesting and could have practical considerations for human clinical use, but I doubt about the practical relevance in poultry rearing.

The introduction is written too general about the control of C. jejuni in poultry and the increasing risk of AMR in this pathogen. As a result, the real focus of this paper which is the effect of (-)-α-pinene on C. jejuni is not well introduced. For example, quorum sensing which is one of the focus points, is only introduced with a small paragraph of 5 lines, but mentioning only one reference related to C. jejuni in a guinea pig abortion model which is not really relevant for poultry.

The discussion is written too much as a review of literature on the effect of other essential oils or plant components on pathogens and on the importance of improving the control of C. jejuni in poultry in general. It’s not enough focused on the explanation and discussion of the own results. For example, there is no hypothesis why (-)-α-pinene has no effect on the ciprofloxacin resistant C. jejuni population in the broiler infection model.

There are still some corrections in English to be made, this should be checked throughout the manuscript.

Other specific remarks:

-L145: for what purpose was the NCTC 11168 luxS mutant used? As negative control?

-L156: explain AB

-L160: is the negative control correct here (V. harveyi suspension)?

-L166 & 172: incorrect numbering of figures (Fig. S2 before S1)

-L175-176: there can’t be two negative controls, the 11168 luxS mutant and fresh MHB?

-L191: explain MHA-SS; details for cloacal swabs. Campylobacter status of chickens was only checked by direct plating, not by enrichment? What was the detection limit?

-L218: plated and incubated

-L258, table 1: host origin of the strains can be shown here

-L275: strains vs. isolates

-L280: also strain W14861 has this behavior

-L311-312: I don’t understand how the results are presented here versus the negative control (luxS mutant)

-L324-328: this is a repetition from M&M

-L341: where are the horizontal lines in the figure?

-L346: in Fig. 2, I only see 3 colonized birds

-L349: idem, only 7 colonized birds?

-L366: what is the exact meaning of “the potential to lower…”, this is scientifically not well formulated; is it statistically significant or only a trend?

-L373-385: this is a repetition from M&M

-L439: macrolides were not included here

Reviewer #2: The subject of the work is interesting and innovative. It raises an important issue regarding the search for alternative methods to combat Campylobacter in poultry. It would also be worth checking (maby in next stage of research) what effect the (-)-a-pinene has on other microorganisms inhabiting the intestines in birds. Does it not inhibit the growth and colonization of beneficial microflora such as lactobacilli or bifidocabterie?

I have some reservations about the methodology of the experiment regarding the assessment of the influence of a-pinene on the colonization of Camylobacter in chicks. The authors took cloacal swabs, diluted in MHB and plated on agar; the result is given in cfu/g feces. How did the authors weigh droppings when they took a swab?

The authors also tend to repeat in the Results chapter information from the Materials and Methods chapter, e.g. P17L324-328; P19L373-377.

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30 Nov 2019

Response to reviewers

Title of manuscript: (-)-α-Pinene reduces quorum sensing and Campylobacter jejuni colonization in broiler chickens

Reviewer #1

1. The paper is largely based on a previous study of the same research groups (Kovac et al. 2015) in which both the antimicrobial activity and the resistance modulatory activities of (-)-α-pinene in C. jejuni against two fluoroquinolones was investigated. In the present paper, the C. jejuni strain collection was extended to confirm these activities, but surprisingly the strain (NCTC 11168) used for inoculation of the chickens was not included.

Answer: The strain used for the chicken inoculation was also used in the in-vitro resistance modulatory part of the study. In Table 1, the last strain under the designation “11168” is the inoculation strain. We have changed this designation to “NCTC 11168” now, to avoid further confusion.

2. In addition, now also the inhibition of quorum sensing was shown in a Vibrio harveyi model. Based on these observations, a broiler chicken infection model was used to see if (-)-α-pinene added in the drinking water would reduce the C. jejuni load in the gut. In contrast to the in vitro results, no resistance modulatory effect was seen in the chickens, but where are the in vitro data which proof this effect for the inoculation strain ?

Answer: Please note that in Table 1, where the in-vitro resistance modulatory data are shown, the data for the inoculation strain (NCTC 11168) are shown in the bottom part. The resistance against ciprofloxacin showed a 2-fold change, and the resistance against erythromycin showed a 4-fold change.

3. No effect, either (-)-α-pinene alone or in combination with enrofloxacin, was seen on the colonization level. Only through a direct gavage of (-)-α-pinene, a statistically significant but very modest reduction (< 1 log cfu/g feces) was observed for the ciprofloxacin sensitive C. jejuni population only. In fact, for half of the chickens infected with this sensitive C. jejuni population, the C. jejuni excretion level was comparable with the untreated control. The statistical significance was probably only obtained through one bird showing a considerable lower C. jejuni level. I therefore doubt about the soundness of the statistical analysis using a t-test and the inclusion of the outlier bird.

Answer: As the data has a normal distribution, which was tested with Kolmogorov-Smirnov and Shapiro-Wilk tests (see Table1), students t-test is the appropriate statistical test here. We have now compared the statistical significance of the data with all of the data pointes (Table 2) and without the outlier (Table3, and the statistical significance, albeit weaker, still remains.

Table 1. Normality tests.

Tests of Normality

Group Kolmogorov-Smirnova Shapiro-Wilk

Statistic df Sig. Statistic df Sig.

CjejuniNo cipS 0.228 10 0.151 0.900 10 0.221

cipS-AP 0.156 10 0.200 0.947 10 0.628

cipR 0.179 10 0.200 0.954 10 0.715

cipR-AP 0.145 10 0.200 0.952 10 0.687

Table 2. T-tests with all of the data.

Levene's Test for Equality of Variances t-test for Equality of Means

CjejuniNo F Sig. t df Sig. (2-tailed) Mean Difference Std. Error Difference 95% Confidence Interval of the Difference

Lower Upper

Equal variances assumed 1.287 0.271 2.397 18 0.028 0.7788 0.325 0.09613 1.462

Equal variances not assumed 2.397 14.26 0.031 0.7788 0.325 0.08307 1.475

Table 3. T-tests without outlier in the FQ-S (CipS) -AP group.

Levene's Test for Equality of Variances t-test for Equality of Means

Nooutlier F Sig. t df Sig. (2-tailed) Mean Difference Std. Error Difference 95% Confidence Interval of the Difference

Lower Upper

Equal variances assumed 0.121 0.732 2.166 17 0.045 0.56984 0.26306 0.01483 1.12485

Equal variances not assumed 2.139 15.302 0.049 0.56984 0.26637 0.00305 1.13662

4. Although the different categories of results are valuable on their own, there is a general lack of coherence between the results. The quorum sensing results have almost no connection with the other types of data and as they are only observed in vitro, there is no indication that this activity will also appear in vivo and therefore would help to find the mechanism of the observed C. jejuni colonization decrease with (-)-α-pinene direct gavage. “

Answer: The Introduction (L62-L63, L81-L88) has been changed and hopefully these changes will clarify and link the quorum sensing better with the rest of the data. We have explored quorum sensing inhibition as one of the possible mechanisms of (-)-α-pinene activity, together with the (previously published) efflux pump inhibition and membrane disruption. We are not proposing that quorum sensing inhibition is solely responsible for C. jejuni reduction after treatment with (-)-α-pinene, but rather that it is one of the actions of (-)-α-pinene against C. jejuni (L436 –L444).

5. The rationale to look for a synergy between (-)-α-pinene and enrofloxacin in broiler chickens is not clear. Enrofloxacin is used in human medicine, but is not used in broilers to prevent or combat a C. jejuni infection. The observation of the enrofloxacin/ciprofloxacin resistance modulation by (-)-α-pinene in vitro is interesting and could have practical considerations for human clinical use, but I doubt about the practical relevance in poultry rearing.

Answer: (-)-α-pinene was shown to modulate C. jejuni resistance to fluoroquinolone antibiotics (Table 1) and was expected to affect the emergence of fluoroquinolone resistant Campylobacter during antibiotic treatment. Thus a rationale to examine a synergy between (-)-α-pinene and enrofloxacin was warranted. We have modified the manuscript to clarify this point. The chicken model is a well established in vivo system to study fluoroquinolone resistance development in Campylobacter, which also occurs in human patients treated with a fluoroquinolone antibiotic. Thus the finding is relevant to human clinical cases where fluoroquinolones are commonly used to treat campylobacteriosis. Additionally, enrofloxacin is still being used in poultry (and other animals) in certain parts of the world for treatment of bacterial infections, such as colibasillosis and Mycoplasma infections. Due to prevalence of C. jejuni in poultry intestinal tract as a commensal, the treatment of poultry with enrofloxacin will result in resistance development in C. jejuni, which can be subsequently transmitted to humans via the food chain. If (-)-α-pinene is found to be effective in inhibiting resistance development in Campylobacter, it may be used as adjunct therapy to control the development of antibiotic-resistant Campylobacter in different hosts. In this study we used only one strain for the in vivo experiment. Additional work needs to be done to examine the potential of (-)-α-pinene.

6. The introduction is written too general about the control of C. jejuni in poultry and the increasing risk of AMR in this pathogen. As a result, the real focus of this paper which is the effect of (-)-α-pinene on C. jejuni is not well introduced. For example, quorum sensing which is one of the focus points, is only introduced with a small paragraph of 5 lines, but mentioning only one reference related to C. jejuni in a guinea pig abortion model which is not really relevant for poultry.

Answer: Thank you for this observation. We have edited the Introduction (L81-L98) and hope that this clarifies better the background and purpose of this study.

7. The discussion is written too much as a review of literature on the effect of other essential oils or plant components on pathogens and on the importance of improving the control of C. jejuni in poultry in general. It’s not enough focused on the explanation and discussion of the own results. For example, there is no hypothesis why (-)-α-pinene has no effect on the ciprofloxacin resistant C. jejuni population in the broiler infection model.

Answer: We have changed some parts of the Discussion for clarification (L381-L383; 431-432: 439-440), although we feel that the comparison of pinene with essential oils and their constituents is necessary as pinene is the major constituent of many essential oils.

We have attempted to explain the lack of effect of pinene on FQ-R strains by the increased fitness of FQ-R strains observed by Luo et al. (2005) (L430-L436). We feel that more speculation about the reason for the weaker effect observed here would be unwise, as we do not have more information at this point.

8. There are still some corrections in English to be made, this should be checked throughout the manuscript.

Answer: The manuscript has been proofread and edited by a professional mother-tongue scientific editor.

9. Other specific remarks:

-L145: for what purpose was the NCTC 11168 luxS mutant used? As negative control?

Answer: Yes, it was used as a negative control. This has now been corrected in the manuscript.

-L156: explain AB

Answer: AB medium is described in the Bacterial strains and growth conditions (L111-L114).

-L160: is the negative control correct here (V. harveyi suspension)?

Answer: No, this was an error, and we apologize for it. It has now been removed from the manuscript.

-L166 & 172: incorrect numbering of figures (Fig. S2 before S1)

Answer: Thank you for this observation. The labeling has been corrected in text, and the Figure labels have remained the same.

-L175-176: there can’t be two negative controls, the 11168 luxS mutant and fresh MHB?

Answer: This is correct, and the error has been corrected. MHB is the “blank” value.

-L191: explain MHA-SS?

Answer: MHA-SS is described in Bacterial strains and growth conditions: L113-L115.

-details for cloacal swabs

Answer: We considered approximately 100 mg of feces per cloacal swab, and we have presented the data accordingly.

This is the method previously used in other studies (e.g., Sahin et al. (2003) doi: 10.1128/AEM.69.9.5372-5379.2003, and Luo et al. (2003) doi:10.1128/AAC.47.1.390-394.2003), and to maintain consistency and for better comparisons of our data with already published data, we would like to keep this as it is. The description has been added into the manuscript for clarification (L215-L220). Cloacal swabs are commonly used to detect Campylobacter shedding in poultry in a non-invasive way, as was used in the present study.

-Campylobacter status of chickens was only checked by direct plating, not by enrichment? What was the detection limit

Answer: Direct plating is considered a better option for Campylobacter spp. isolation from chicken feces, compared to enrichment (Musgrove et al., 2001; Sahin et al., 2003). The detection limit was 100 CFU/g feces. The description of the method has been adjusted for better clarification (L215-L220).

Musgrove, M.T., M.E. Berrang, J.A. Byrd, N.J. Stern, and N.A. Cox. 2001. Detection of Campylobacter spp. in ceca and crops with and without enrichment. Poult Sci. 80:825–828.

Sahin, O., Q. Zhang, and T.Y. Morishita. 2003. Detection of Campylobacter. In: Microbial Food Safety in Animal Agriculture. M.E. Torrence and R.E. Isaacson, eds. Iowa State Press, Ames, IA.183–193

-L218: plated and incubated

Answer: The text has been added to L218-L219.

-L258, table 1: host origin of the strains can be shown here

Answer: The description has been added as suggested.

-L275: strains vs. isolates

Answer: Corrected.

-L280: also strain W14861 has this behavior

Answer: Thank you for the observation. This has been corrected (L285).

-L311-312: I don’t understand how the results are presented here versus the negative control (luxS mutant)

Answer: The error was corrected for L314-317.

-L324-328: this is a repetition from M&M

Answer: Corrected.

-L341: where are the horizontal lines in the figure?

Answer: In each stack of data points there is a (small) horizontal line that represents the means of each dataset. Due to some technical difficulties with the Figure format, this was not presented properly. This has now been corrected.

-L346: in Fig. 2, I only see 3 colonized birds

Answer: Four birds are colonized, but the symbols are overlapping. The symbol with the thicker edges (the lowest) is actually two symbols. This could not be corrected on this graph.

-L349: idem, only 7 colonized birds?

Answer: As in the previous point, here we have two symbols on the upper part that are overlapping, and thus look like one symbol with thicker edges.

-L366: what is the exact meaning of “the potential to lower…”, this is scientifically not well formulated; is it statistically significant or only a trend?

Answer: This has been corrected.

-L373-385: this is a repetition from M&M

Answer: Corrected.

-L439: macrolides were not included here

Answer: Corrected. The sentence has been rearranged for clarification.

Reviewer #2

1. The subject of the work is interesting and innovative. It raises an important issue regarding the search for alternative methods to combat Campylobacter in poultry. It would also be worth checking (maby in next stage of research) what effect the (-)-a-pinene has on other microorganisms inhabiting the intestines in birds. Does it not inhibit the growth and colonization of beneficial microflora such as lactobacilli or bifidocabterie?

Answer: Thank you for this observation. Any treatment added to chicken water that can affect C. jejuni numbers will probably also influence other members of the intestinal microbiota, although it does not necessarily mean a negative influence on positive microbes, as are bifidobacteria. This is something that will indeed be considered for future work.

2. I have some reservations about the methodology of the experiment regarding the assessment of the influence of a-pinene on the colonization of Camylobacter in chicks. The authors took cloacal swabs, diluted in MHB and plated on agar; the result is given in cfu/g feces. How did the authors weigh droppings when they took a swab?

Answer: Each swab diluted was considered to have approximately 100 mg of fecal matter and was diluted in 1 mL of MHB so as to get a 10-fold dilution. Dilutions were plated and data are shown as cfu/g feces, with a detection limit of 100 cfu/g feces. We considered 100 mg of feces per cloacal swab and presented the data accordingly.

This is the method previously used in other studies (e.g., Sahin et al. (2003) doi:10.1128/AEM.69.9.5372-5379.2003 and Luo et al. (2003) doi:10.1128/AAC.47.1.390-394.2003, and for consistency and better comparisons of our data with already published data we prefer to keep this as it is. A description has been added into the manuscript for clarification (L209-L212). Cloacal swabs are commonly used to detect Campylobacter shedding in poultry in a non-invasive way, and it was used in the present study as such.

3. The authors also tend to repeat in the Results chapter information from the Materials and Methods chapter, e.g. P17L324-328; P19L373-377.

Answer: Corrections have been made to these parts of the text.

Editor

As is often the case, the two reviewers have highlighted different points in their reviews. I would urge the authors to read these comments carefully for any revision they make, as there are quite a few of them. However, in terms of what needs to be addressed, the following four points can be made:

1) Methodological issues - sampling as swabs, but reporting data as if it were faeces, and concerns over the impact of outlier data in the conclusions drawn

Answer: Corrections and additional explanations have been added into the text to explain the swabbing process better.

Additional statistical analysis was performed using all of the data and the data without the outlier, and the statistical significance remained. So we believe that the effect of pinene shows real significance.

2) Repetition of methods and results - both reviewers comment on this

Answer: This has been corrected.

3) Justification for issues around the study design - relevance of the study to humans in terms of drug use in humans and chickens

Answer: Corrections have been made to the text to clarify this matter. We do not propose that enrofloxacin should be used for Campylobacter control in poultry, and we apologize for the confusion.

4) Discussion - some clarification over aspects in regards to framing the results in the current literature

Answer: Some modifications have been made throughout the Discussion (L395-L398; 435-443: 486-488).

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Answer: Corrections have been made throughout the manuscript.

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Answer: Additional details in the method section have been added (L184-L185).

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Answer: DOIs for all of the data are listed in the data availability statement together with links that reviewers can use to access the data before it is published. DOIs will be activated upon manuscript acceptance. Appropriate DOIs (but still inactive) are also listed below.

Data availability:

Figure 1.

Doi: 10.6084/m9.figshare.8234153

Figure 2.

Doi: 10.6084/m9.figshare.8234192

Figure 3.

Doi: 10.6084/m9.figshare.8234189

Figures S1 and S2

Doi: 10.6084/m9.figshare.8234156

Tables S1 and S2.

Doi: 10.6084/m9.figshare.8234159

Submitted filename: Simunovicetal2019_Response to Reviewers.docx

Click here for additional data file.

2 Mar 2020

(-)-α-Pinene reduces quorum sensing and Campylobacter jejuni colonization in broiler chickens

PONE-D-19-20048R1

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Reviewer #1: Yes: Marc Heyndrickx

Reviewer #3: Yes: Xiaonan Lu

12 Mar 2020

PONE-D-19-20048R1

(-)-α-Pinene reduces quorum sensing and Campylobacter jejuni colonization in broiler chickens

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