In vitro antibacterial activity of thymol and carvacrol and their effects on broiler chickens challenged with Clostridium perfringens

Background

Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium, which is usually classified into five toxinotypes (A, B, C, D, and E) according to the production of four major toxins, called alpha, beta, epsilon and iota. Although C. perfringens is a commensal bacterium of the intestine, C. perfringens type A is considered the main causative agent of necrotic enteritis (NE) in poultry [1, 2]. Proliferation of pathogenic C. perfringens and released toxins, especially NetB toxin, result in NE in poultry [2, 3]. NE is a widespread disease in poultry, as estimated to cost the international poultry industry approximately two billion US dollars annually [4]. Impaired feed digestion and absorption, reduced growth rate, and mortality are the major reasons for production losses associated with NE [2, 5].

The use of in-feed antibiotics has been the main strategy for controlling NE in poultry. However, public concern about the threat of antibiotic-resistant pathogens has forced the poultry industry to consider alternatives to antibiotics for poultry production [5]. Probiotics, prebiotics, organic acids, enzymes, and essential oils (EO) are among the alternatives [5]. Also, because of consumer preference for natural products, the use of EO has increased appeal [6].

The antibacterial properties of EO have long been recognized and widely tested in vitro against a wide range of pathogenic bacteria, including both Gram positive and Gram negative bacteria [6-8]. Although the antibacterial mechanism of EO and their constituents is not fully understood, studies have shown that constituents with a phenolic structure, such as eugenol, carvacrol and thymol have the greatest bactericidal activities, followed by aldehydes, ketones, alcohols, ethers and hydrocarbons [9-11]. However, it seems that the efficacy of EO is not consistent in vivo: both improved and unchanged growth performance and intestinal microbiota have been reported in pigs and chickens [6, 8, 12-14]. Actually, it is difficult to compare the efficacy of EO considering the fact that EO blends containing various constituents have been used in vivo in published reports [6, 8, 12-15]. In addition, little information is available regarding the relationship between antibacterial activities in vitro and in vivo. Therefore, the aim of the present study was to investigate the efficacy of an EO product as well as its components thymol and carvacrol on pathogenic bacteria and benefical Lactobacillus strains, and to investigate the effects of EO on broiler chickens challenged with C. perfringens. In addition, thymol and carvacrol concentrations in intestinal digesta were assayed.

Methods

In vitro study

Chemicals

Thymol and carvacrol were purchased from Sigma-Aldrich Corporation (St Louis, MO, USA), with compound purities at?~?98 %. A commercial EO product was provided by Novus International Inc. (St Charles, MO, USA), which contained 25 % thymol and 25 % carvacrol as active components, 37 % silicon dioxide as a caking inhibitor, and 13 % glycerides as stabilizing agents.

Preparation of cultures

Thymol, carvacrol and the commercial EO product were individually tested against a panel of undesirable bacteria and beneficial Lactobacillus strains. The undesirable bacteria included two chicken Escherichia coli field strains CVCC1553 and CVCC1490 (serotype O78), chicken C. perfringens field strains (CVCC2027 and CVCC2030), Salmonella enterica serovar Typhimurium (CVCC541), Salmonella enterica serovar Enteritidis (CVCC2184) and Salmonella enterica serovar Pullorum (C79-13), which were obtained from China Veterinary Culture Collection Center (Beijing, China). Another S. Enteritidis field strain (ATCC13076) was obtained from American Type Culture Collection (Manassas, VA, USA). In addition, three strains of Lactobacillus were tested. L. acidophilus (GIM1.730) was obtained from Guangdong Microbiology Culture Center (Guangdong, China), and L. reuteri and L. salivarius were isolated from the gastrointestinal content of healthy broiler chickens by our laboratory staff.

Overnight cultures of E. coli and Salmonella were prepared freshly by cultivation from frozen stock at 37 °C in Luria-Bertani broth with continuous shaking. Cultures of C. perfingens were prepared anaerobically at 37 °C overnight in cooked meat medium and Man-Rogosa-Sharpe (MRS) broth used for growing of Lactobacillus at 37 °C without shaking.

Minimum inhibitory concentration and minimal bactericidal concentration assay

The minimum inhibitory concentration (MIC) was determined via a conventional broth dilution method, as described by Ivanovic et al. with some modifications [16]. Briefly, the commercial EO product, thymol, and carvacrol were initially dissolved as 300 mg/mL stock solution in dimethylsulfoxide (DMSO) and diluted to 6 mg/mL in Mueller-Hinton broth with vigorous shaking. Then, two-fold serial dilutions of thymol and carvacrol were prepared, producing concentrations at 6000, 3000, 1500, 750, 375, 187.5, 93.75 and 46.875 ?g/mL. For the detection of Lactobacillus, MRS broth was used for dilution. In order to avoid the antibacterial effects of DMSO itself, the final DMSO concentrations never exceeded 2 % (by volume). Then, each diluted broth was inoculated with fresh microbial suspension (final concentration: 1?~?5?×?105 colony-forming units (cfu)/mL). E. coli and Salmonella were incubated at 37 °C with continuous shaking overnight. C. perfringens and Lactobacillus were incubated anaerobically at 37 °C for 24 and 16 h, respectively, without shaking. A positive growth control containing 2 % DMSO without EO or components, and a negative control containing no bacteria were included in each experiment. After incubation, the optical density (OD) of suspension was measured using a spectrophotometer at 595 nm. In addition, 50 ?L from each broth dilution was inoculated for enumeration in duplicate onto nutrient agar for E. coli and Salmonella, sulfite-polymyxin-sulfadiazine agar for C. perfringens, and MRS agar for Lactobacillus, all by the spread plate method. MIC was defined as the lowest concentration of EO or components that showed no increase in OD following incubation. The minimal bactericidal concentration (MBC) was defined as the lowest concentration of EO or components with which no viable bacteria were detected. All assays were performed in triplicate.

Combination assay

A combination assay between thymol and carvacrol was performed by the checkerboard method, as previously described [17]. Two-fold serial dilutions of one component were tested in the presence of serial concentrations of the other component (which did not inhibit bacterial growth alone). The fractional inhibitory concentration (FIC) was calculated as follows: FIC of component A??=?MIC of component A in combination divided by the MIC of component A alone, FIC of component B??=?MIC of component B in combination divided by the MIC of component B alone, and FIC index?(FICI)?=?FIC of component A?+?FIC of component B. An FICI?<?0.5 was considered to demonstrate synergy. When an FICI fell between 0.5 and 1.0, it was defined as an additive effect and, between 1.0 and 4.0, it was classified as no interaction. Finally, an FICI?>?4.0 indicated antagonism between the components in a combination.

In vivo study

Birds, diets, and experimental design

All experimental procedures were approved by the China Agricultural University Animal Care and Use Committee. A total of 448 one-day-old male broiler chicks (Cobb 500) were used for a 28-day experiment. Chicks were assigned to eight treatments, following a 4?×?2 factorial arrangement in a randomized complete block design to evaluate dietary EO supplementation (0, 60, 120, or 240 mg/kg EO in wheat-based diet), pathogen challenge (with or without oral gavage of C. perfringens from day 14 to 21) and their interactions. Each treatment consisted of eight replicate pens (seven birds/pen). The EO used in this trial was the commercial product mentioned above, which contained 25 % thymol and 25 % carvacrol as active components. Chickens were fed starter (day 0-21) and finisher (day 21-28) diets in the form of mash and had access to feed and water ad libitum. Proliferation of C. perfringens was promoted by formulating antibiotic-free and coccidiostat-free wheat-based diets. All nutrients were formulated to meet or exceed the feeding standard of China (NY/T 2004) for broilers [18] (Table 1). [ Table Omitted - see PDF ]

Table 1

Diet composition and nutrient levels

Item (%, unless otherwise indicated)

Starter diets (day 0-21)

Grower diets (day 22-28)

Ingredient

Wheat

62.75

68.5

Soybean meal

29.61

23.72

Soybean oil

3.40

4.00

Dicalcium phosphate

1.91

1.63

Limestone

1.04

0.96

Sodium chloride

0.35

0.35

Choline chloride (50 %)

0.25

0.25

L-Lysine (99 %)

0.25

0.24

DL-Methionine (98 %)

0.19

0.11

Antioxidants

0.03

0.03

?Trace mineral premix a

0.20

0.20

?Vitamin premix b

0.02

0.02

Calculated nutrient levels

Metabolizable energy (Mcal/kg)

2.90

2.98

Protein

21.00

19.00

Calcium

1.00

0.90

Available phosphorus

0.45

0.40

Lysine

1.15

1.00

Methionine

0.50

0.40

a The trace mineral premix provided the following (per kilogram of diet): manganese, 100 mg; zinc, 75 mg; iron, 80 mg; copper, 8 mg; selenium, 0.25 mg; iodine, 0.35 mg

b The vitamin premix provided the following (per kilogram of diet): vitamin A, 18750 IU; vitamin D3, 3750 IU; vitamin E, 28 IU; vitamin K3, 3.975 mg; thiamine mononitrate, 3 mg; riboflavin, 9 mg; vitamin B12, 0.0375 mg; d-biotin, 0.150 mg; folic acid, 1.875 mg; d-calcium pantothenate, 18 mg; nicotinic acid, 75 mg

Clostridium perfringens challenge and sampling

C. perfringens challenge was conducted as originally developed by Dahiya et al. [19]. The particular organism, CVCC2027, was a type A field strain, isolated from a clinical case of NE in chickens, which did not carry the NetB gene, as determined by polymerase chain reaction (PCR). Briefly, the organism was cultured anaerobically on tryptose-sulphite-cycloserine agar base at 37 °C for 18 h, and then aseptically inoculated into cooked meat medium and incubated anaerobically at 37 °C overnight. All birds in challenged groups were orally gavaged in the crop once per day with 1.0 mL of actively growing C. perfringens culture from day 14 to 20 (1.0?×?108 cfu/mL). On day 21 and 28, one bird per replicate was randomly selected and killed by intracardial administration of sodium pentobarbital (30 mg/kg body weight) and jugular exsanguination prior to sample collection.

Intestinal lesion score

The small intestine from each bird was opened and scored blindly on a scale from zero to four as described by Dahiya et al. [19]: 0?=?normal intestinal appearance with no lesion, 0.5?=?severely congested serosa and mesentery engorged with blood, 1?=?thin walled and friable intestines with small red petechiae (>5), 2?=?focal necrotic lesions, 3?=?patches of necrosis (1 to 2 cm-long), and 4?=?diffused necrosis typical of field cases.

Bacteriological examination

On day 21 and day 28, digesta for bacteriological examination were collected aseptically from ileum (from ileum midpoint to 2 cm proximal to ileocecal junction) and caecum, and stored at ?80 °C. Bacterial populations were detected by the method of absolute quantitative real-time PCR (RT-PCR), as described by Wise and Siragusa, with some modifications [20]. Genomic DNA was isolated from 200 mg of digesta from ileum and caecum using a commercial kit (QIAamp DNA Stool Mini Kit, Qiagen Inc., Valencia, CA, USA). Extracted DNA was stored at ?20 °C until analysis.

Standard curves for RT-PCR were prepared using DNA extracted from pure cultures to produce a high concentration of the target DNA by normal PCR amplification. Primer sequences were used in previous studies, which were designed on the basis of 16s rDNA sequences [21-23]. Target groups, primer sequences, amplicon sizes, and references are shown in Table 2. The targeted Escherichia subgroup contained genera of E. coli, Hafnia alvei and Shigella [22]. E. coli competent cells DH5? (Takara Bio Inc., Japan) were used to create plasmid standards. Firstly, PCR products were purified using a PCR purification kit (Biomed Gene Technologies, Beijing, China), and then cloned into pCR®2.1 using a TA cloning kit (Invitrogen Corporation, Carlsbad, CA, USA), following the manufacturer's protocol. Purified insert-containing plasmids were quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the number of target gene copies was calculated by the following formula according to Lee et al. [24]: [ Table Omitted - see PDF ]

Table 2

16s rDNA real-time PCR primers used to quantify intestinal bacteria

Target

Primer sequence (5?-3?) a

Amplicon size (bp)

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