The C. jejuni NCTC11168 was kindly provided by Chinese Center for Disease Control. C. jejuni strains were routinely cultured on Mueller-Hinton (MH) agar (Oxoid, Basingstoke, UK) or blood agar plates at 42°C under microaerophilic conditions. E. coli DH5α was grown aerobically in Luria-Bertani medium at 37°C. The Ham’s F-12 nutrient powder mixture (SH30010, Thermo Fisher, Waltham, MA, USA) and Ham’s F-12m nutrient powder mixture (RR13033.01, Thermo Fisher, Waltham, MA, USA) lacking leucine were used for leucine biosynthesis test.

A 2.5-Kb fragment containing Cj1199 and its flanking region was amplified with primers of cj1199F and cj1199R (Table 1) by PCR and cloned into pGEM-T easy vector (Promega, Madison, WI, USA) to yield pL1199. A chloramphenicol resistance cassette (Cm^r marker) was amplified from pUOA18 using primers of ecatF and ecatR. Primers rF and rR were used to inversely amplify pL1199. The PCR product of the inverse amplification of pL1199 was digested with SmaI and BamHI and ligated with the Cm^r marker to yield plasmid pL1199T. The plasmid pL1199T was transformed into E. coli DH5α and then introduced to C. jejuni NCTC11168 by natural transformation (Jeon et al., 2010). The Cj1199 deletion mutant (ΦCj1199) was selected on MH agar containing chloromycetin (20 μg/ml) and confirmed by PCR and DNA sequencing.

The cells of ΦCj1199 or C. jejuni NCTC11168 were grown in MH agar plate for 24 h at 42°C under microaerophilic conditions. For RNA extraction, RNAprotect Bacteria Reagent (Qiagen, Valencia, CA, USA) was added to the cultures immediately after the incubation to stabilize mRNA. The total RNA from each sample was extracted using the RNeasy Protect Mini Kit (Qiagen, Valencia, CA, USA) following the manufacture’s protocol. RNA samples were extracted from four independent experiments.

The differential gene expression between ΦCj1199 and C. jejuni NCTC11168 was identified using DNA microarray which was supplied by CapitalBio Corporation (Santa Clara, CA, USA). Briefly, iScript cDNA synthesis kit (BioRad, Hercules, CA, USA) was used for synthesis of cDNA from an RNA template via reverse transcription. The cDNA was then labeled with Cy5 or Cy3 dye. The labeled cDNA probes were co-hybridized onto one microarray slide (Roche NimbleGen 4 × 72K, Indianapolis, IN, USA). Hybridized slides were scanned using NimbleGen MS200, and the fluorescence intensities were collected with NimbleGen Scan Software. The linear normalization method was used for data analysis based on expression of the housekeeping genes. Normalized data was log transformed and loaded into MANOVA under R environment. Microarray spots with false discovery rate (FDR)-corrected q-values < 0.01 and fold change ≥2 in the T-test were regarded as differentially expressed genes. The differentially expressed genes were classified based on the genomic annotation in NCBI and then subjected to KEGG database for pathway analysis.

The surplus cDNA of ΦCj1199 and C. jejuni NCTC11168 was subjected to real-time quantitative reverse transcriptase PCR (qRT-PCR) analysis to confirm the expression of some respective genes identified by DNA microarray. The primers (Table 1) of respective genes were designed using Primer 5 software. The qRT-PCR was performed using SYBR Green Ex Taq^TM kit (Takara, Madison, WI, USA) in IQ5 Multicolor Real-time PCR Detection System (Bio-Rad) following method described in previous study (Hao et al., 2010). The qRT-PCR was initiated with a 30 s denaturation at 95°C, followed by 38 cycles of amplification with 5 s of denaturation at 95°C, 30 s of annealing according to the melting temperatures of amplifications. The melting curve was performed from 65 to 95°C (1 s hold per 0.2°C increase) to check the specificity of the amplified product.

Minimum inhibitory concentrations (MICs) of azithromycin (AZM), erythromycin (ERY), tylosin (TYL), ciprofloxacin (CIP), nalidixic acid (NAL), olaquindox (OLA), florfenicol (FFC), doxycycline (DOX), gentamicin (GEN), and levofloxacin (LEV) against ΦCj1199 and C. jejuni NCTC11168 were determined with broth microdilution method following the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2006). The concentrations of the antimicrobial agents tested ranged from 0.01 to 16 μg/mL. The microdilution plates (96 well plates) were incubated at 42°C for 24 h and the MIC was determined as the lowest concentration where visible bacterial growth was inhibited at the end of the incubation period. C. jejuni ATCC 33560 was used as a quality control strain.

For the minimal bactericidal concentration (MBC) of drugs against C. jejuni strains, 100 μL from each well was successively diluted 1:10 in MH broth. Then 10 μL was spread on MH agar plates and incubated at 42°C for 24 h for colony forming unit (CFU) counting. The MBC was defined as the lowest drug concentration that resulted in a 99.9% reduction in the bacterial density. The final result was expressed as mean of five independent experiments.

The mutant prevention concentration (MPC) of drugs was determined by agar method (Blondeau et al., 2001). The inoculum of C. jejuni was concentrated to 10¹⁰ CFU/mL. Bacterial suspensions were inoculated on the agar plates containing serial dilutions of drugs and cultured for 96 h. The MPC was the lowest drug concentration on agar plates without bacterial growth under micro-aerobic conditions.

The concentration of bacteria was adjusted to a 0.5 McFarland density. Serial twofold dilutions of erythromycin were prepared with MH broth. The final concentrations of erythromycin ranged from 0.0625 to 8 μg/mL. A 100 μL aliquot of bacteria was added to 900 μL MH broth containing different concentrations of erythromycin and incubated at 42°C in a micro-aerobic condition for 3 h. Each dilution was then centrifuged and the supernatant was discarded. The pellet was washed twice with sterile PBS and propidium iodide (PI), a membrane-impermeable DNA-intercalating dye, was used to stain the erythromycin-treated bacterial cells. One milliliter of buffer and 5 μL of PI were added to each sample, and then incubated for 30 min at 4°C. FCM analysis was conducted on CyAn ADP^TM FC500 flow cytometer with Summit^TM software (Beckman Coulter, Miami, FL, USA). The intensity of fluorescence of 10,000 cells labeled with PI was analyzed to obtain the mean channel fluorescence (MCF).

ΦCj1199 and NCTC11168 were subjected to step-wise selection in MH agar plate containing erythromycin. In the first step of selection, cells were plated on MH agar containing 0.25 μg/mL erythromycin. After 3–5 days of incubation under microaerophilic condition at 42°C, colonies were selected and repeatedly incubated with same concentration of erythromycin up to five times. The mutants obtained from first-step selection were then subjected to next step selection by exposing the cells to a twofold increased concentration of erythromycin. The procedure of selection was repeated up to 10 times to obtain highly erythromycin-resistant mutants. All in vitro-selected mutants were subjected to MIC test using the broth microdilution method as described above.

Growth curves of ΦCj1199 and C. jejuni NCTC11168 were measured individually and repeated three times. Briefly, a fresh C. jejuni culture was diluted 100-fold into 10 mL MH broth and incubated for up to 36 h. Viable C. jejuni counts were determined at 4, 12, 24, and 36 h.

Pair-wise competition experiments were used to estimate the growth rate of ΦCj1199 compared with C. jejuni NCTC11168 in the erythromycin containing (1/2 MIC) or drug-free MH broth. Briefly, the same densities of the parent and mutant cultures were prepared before experiments. Equal volumes (0.1 mL) of adjusted cultures were mixed into 10 mL of fresh medium with or without erythromycin. After 24 h incubation, 0.1 mL bacterial culture was then transferred to another 10 mL fresh medium. The passage was repeated up to 10 times and the initial and final concentrations of parent and mutant were determined by standard plate counting on MH plate with (for the mutant population) or without 10 μg/mL chloromycetin (for the total population), respectively. The assays were repeated three times with three technical replicates.

Cultures of ΦCj1199 and C. jejuni NCTC11168 were adjusted to the same densities at the beginning of the experiments. Equal volumes (0.1 ml) of adjusted cultures were added into 10 mL Ham’s F-12 and F-12m individually. After 48 h incubation under microaerophilic conditions at 42°C, 0.1 mL of bacterial culture was then transferred to another 10 mL of fresh medium. The passage was repeated up to five times. Viable C. jejuni counts were determined by dilution and spatula method on MH plates (ICC, 1992). The experiment was performed in triplicate.

The 20 genes down-regulated in ΦCj1199 are shown in Table 2. Five genes (leuA, leuB, leuC, leuD, and trpE) were involved in amino acid biosynthesis. The leuABCD genes participated in leucine biosynthesis pathway. Five genes (Cj0919c, Cj0920c, peb1A, pebC, and ceuC) encoded transporters or binding proteins. The permease protein (Cj0919c-Cj0920c), substrate-binding protein (Peb1A), and ATP-binding protein (PebC) worked cooperatively in an ABC-type amino acid transport system. The ceuC encoded enterochelin uptake permease which was also transporters or binding protein. The Cj0246c was involved in signal transduction. Six genes (Cj0560, Cj1356c, Cj0423, Cj0424, Cj0425, and Cj0200c) encoded putative integral membrane or periplasmic proteins. Another three (Cj1025c, Cj1722c, and Cj1388) were hypothetical genes.

Only Cj1241 gene encoding a putative major facilitator superfamily (MFS) transport protein was up-regulated in ΦCj1199. Using qRT-PCR to verify the microarray results, Cj1241 gene was confirmed to be up-regulated, while other four respective genes (Cj0920c, Cj1388, Cj0560, and Cj1717c) were confirmed to be down-regulated (Figure 1).

The antimicrobial susceptibility of the parent and mutant strains are presented in Table 3. There was no significant difference between the MIC of 11 antimicrobials in the ΦCj1199 and its parent strain NCTC11168. Both of the strains exhibited very low MIC (MIC ≤ 0.5 μg/mL) to the 14, 15, and 16-membered ring macrolides (erythromycin, azithromycin, and tylosin). The MBC and MPC of five antimicrobials in ΦCj1199 were similar (erythromycin, azithromycin, tylosin, olaquindox, and cydox) with that in NCTC11168 (Table 3).

The erythromycin tolerance of ΦCj1199 to increased concentration of erythromycin was detected by flow cytometer analysis. As shown in Figure 2, the mean value of the fluorescence intensity was increased following the increase of erythromycin concentration. However, when the concentration of erythromycin reached to their MIC (0.125 μg/mL), the mean PI intensity of C. jejuni NCTC11168 was significantly decreased, while the mean PI intensity of ΦCj1199 kept at its highest level. Under the treatment of different concentrations of erythromycin, the mean PI intensity of ΦCj1199 was higher than that of parent strain, which indicated that ΦCj1199 had higher mortality and lower tolerance to erythromycin.

There was no significant difference between ΦCj1199 and NCTC11168 in the first and second steps of selections. When transferred to third step selection by 1 μg/mL erythromycin, ΦCj1199 descendants exhibited less growth than NCTC11168 descendants. When transferred to fourth step of selection, ΦCj1199 descendants could not grow on MH agar containing 2 μg/mL erythromycin, while C. jejuni NCTC11168 descendants grew well and successfully promoted to fifth step of selection by 4 μg/mL erythromycin. The erythromycin MIC of mutants selected from NCTC11168 was equal to or higher than 128 μg/mL, which was significantly higher than erythromycin MIC (4 μg/mL) of mutants selected from ΦCj1199.

The ΦCj1199 and NCTC11168 were separately cultured in macrolide-free MH broth. No significant difference in growth rates was observed between the two strains in single cultures (Figure 3A).

When ΦCj1199 and NCTC11168 were mixed in pairs into drug-free MH broth, population of ΦCj1199 was decreased after the fifth passage and reduced by twofolds of magnitude after the 10th passage (Figure 3B).

In the mixture culture containing 1/2 MIC erythromycin, population of ΦCj1199 was reduced during passages. The ΦCj1199 was outcompeted by NCTC11168 in the drug-containing culture after the fourth passage (Figure 3C).

There is no significant difference of the growth between ΦCj1199 and NCTC11168 in the medium F-12. Both of them grew well after five passages in the medium F-12 (Figure 4). However, the number of ΦCj1199 was remarkably decreased during the five times of passage in the medium F-12m. After the fifth passages in the medium F-12m, the number of ΦCj1199 mutant had been reduced by two orders of magnitude comparing with its parent strain NCTC11168 which kept at the level of 10⁵ CFU/mL. Conclusively, in absence of leucine, the growth of ΦCj1199 was much slower than NCTC11168.