The C. jejuni NCTC11168 (designated as S) was kindly provided by Chinese Center for Disease Control and Prevention. C. jejuni strains were routinely cultured in Mueller-Hinton (MH) medium at 42°C under microaerobic conditions (5% O₂, 10% CO₂, and 85% N₂) in the anaerobic incubator (YQX-II, Shanghai, China) (Mace et al., 2015). The Escherichia coli DH5α was grown aerobically in Luria-Bertani medium at 37°C. The erythromycin-resistant C. jejuni strain 68-ER (designated as R) was descendant of C. jejuni NCTC11168 resulting from in vitro step-wise selection by erythromycin. Plasmids pGEM-T (Promega, Madison, WI, USA) and pMW10 was kindly provided by China Agricultural University and used for mutant vector construction.

The DNA fragment containing Cj0440c gene and its flanking regions was amplified from C. jejuni NCTC 11168 genome using Pfu polymerase (Promega) with primers of Cj0440cF₂ and Cj0440cR₂ (Table 1) and was cloned into pGEM-T easy vector (Promega,) to generate plasmid pCJ0440c. Primers pCj0440cU and pCj0440cL (Table 1) carrying endonuclease restriction sites of KpnI and XbaI were used to inversely amplify DNA fragment from the vector of pCJ0440c using Taq and Pfu polymerase (8:1). A kanamycin resistance cassette (kan) was amplified from pMW10 plasmid with Pfu polymerase (Promega) using primers KanF and KanR (Table 1) which have the same restriction sites of KpnI and XbaI. The amplified DNA fragments of inverse PCR and kan were digested with KpnI and XbaI and purified with a PCR clean-up kit (Generay, Shanghai, China). The digested inverse PCR product was ligated to the kan cassette using T4 DNA ligase (Takara, Dalian, China) to obtain the construct plasmid pCJ0440c-Kan, which was then transformed into E. coli DH5α. The purified plasmid of pCJ0440c-Kan was introduced into S and R via electroporation according to the method described previously (Jeon et al., 2011). Insertional mutants, named SM and RM, respectively, were selected on MH agar plates with 25 μg/ml kanamycin and 50 μg/ml ampicillin. Both PCR and sequencing analysis of the Cj0440c mutants (SM and RM) confirmed that the mutation resulted in deletion of 200 bp of coding sequence in Cj0440c and simultaneous insertion of the kan gene into the same location.

The transcriptional difference between Cj0440c mutants and their parental strains (SM&S and RM&R) was examined by microarray (CapitalBio Corporation, Shanghai, China). Briefly, the strains were separately grown in MH broth for 24 h at 42°C under microaerophilic conditions with shaking. Immediately after the incubation, twice volume of RNA protective reagent (Qiagen, Germantown, MD, USA) was added to the culture (with same OD₆₀₀ of 0.3) to stabilize mRNA. The total RNA from each sample was extracted using RNeasy Protect Mini Kit (Qiagen) and purified using Nucleo^®Spin RNA clean-up kit (Macherey-nagel, Germany). The RNA quality and quantity was determined by formaldehyde denatured gel electrophoresis and Nanodrop^TM 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was synthesized from the extracted RNA using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). The cDNA was labeled by Cy5 or Cy3 dye and co-hybridized onto one microarray slide (NimbleGen 4 K × 72K), scanned by Axon Instruments Gene Pix 4000B (Union City, CA, USA) and read by Gene Pix Pro 6.0 (Axon Instruments). Microarray data were analyzed using Array Star software. The genes with False Discovery Rate (FDR)-corrected q-values < 0.01 and fold change >2 were selected as differentially expressed genes and subjected to NCBI gene annotation, KEGG pathway analysis and STRING 9.05 protein network analysis.

The microarray data obtained in this study have been deposited in the NCBI Gene Expression Omnibus database¹ and assigned accession number GSE49255 and GSE49256.

The same batches cDNA of Cj0440c mutants (SM and RM) and their parental strains (S and R) used in microarray were subjected to qRT-PCR analysis to confirm the transcriptional difference of some respective genes identified by microarray following method described in previous study (Hao et al., 2010). Briefly, the PCR amplification was performed in IQ5 Multicolor Real-time PCR Detection System (Bio-Rad). The cycling conditions were as follows: 3 min of pre-incubation at 95°C, followed by 30 cycles of 10 s at 95°C and 40 s at 60°C. The primer sets used for specific genes are listed in Table 1. 16S rDNA was used as an internal control for normalization. The experiment was done in triplicate to obtain the average value of fold change. The student’s t-test was performed to analyze the significant difference between mutants and their parental stains.

Minimum inhibitory concentrations (MICs) of nine antimicrobial agents (azithromycin, erythromycin, tylosin, ciprofloxacin, olaquindox, chloromycetin, tetracycline, gentamicin, and ceftriaxone) were determined using agar dilution method recommended by Clinical and Laboratory Standards Institute (CLSI). C. jejuni ATCC 33560 was used as a quality control strain.

The presence and length of flagella on the four C. jejuni strains (S, SM, R and RM) were examined using transmission electron microscopy according to a previously described method (Barrero-Tobon and Hendrixson, 2014; Matsunami et al., 2016). Briefly, bacterial suspensions were obtained after washing plate with 2 ml sterile phosphate-buffered saline and spotted on carbon-coated copper grids. The cells were stained with 2% phosphotungstic acid (pH 6.7) for 1 min. Samples were observed employing a HITACHI H-7650 transmission electron microscope (Hitachi, Japan).

To compare the growth kinetics of the mutants with that of the parental strains, a fresh culture (100 μL) of each C. jejuni strain (0.5 McFarland) was inoculated into 100 mL MH broth and the cultures were incubated at 42°C under microaerobic conditions for 36 h with shaking. The growth kinetics was determined by measuring the absorbance in 600 nm (OD₆₀₀) at 0, 4, 8, 12, 20, 22, 24, 27, 31, 33, and 36 h post-inoculation.

Newly hatched broiler chickens (White Leghorns) were purchased from Zhengda Limited Company (Wuhan, China). All the broiler chickens were examined for C. jejuni to ascertain that birds are free of C. jejuni prior to infection all the chickens (Hao et al., 2015).

These chickens were randomly assigned to seven groups with 6 to 10 chickens per group. One group was used as a control. Four groups were used for single colonization test in which 10⁹ CFU C. jejuni strains (S, SM, R and RM) were individually inoculated via oral gavage into each group. Another two groups were used for pairwise competition test in which 10⁹ CFU C. jejuni pairwise mixtures (S&SM or R&RM) were inoculated via oral gavage to each group. Fecal samples were collected from each bird at 3, 6, 9, and 12 days’ post-infection. The CFU of S, SM, R and RM were determined using Campylobacter selective CCDA agar (Oxoid, Thermo Fisher Scientific, Waltham, MA, USA) with or without 25 μg/ml Kanamycin or 50 μg/ml erythromycin. Each sample was spread onto three respective selective plates to obtain the average CFU.

The significance of differences between mutant and parental strain in colonization at each sampling time point was determined by using Student’s t-test, Welch’s t-test to allow for non-constant variation across treatment groups, and the Wilcoxon rank-sum test to allow for non-normality (Guo et al., 2008; Luangtongkum et al., 2012; Xia et al., 2013). Differences were considered significant at a P-value of <0.01.

The animal study was approved by Animal Ethics Committee of Huazhong Agricultural University (HZAUCH 2013-002) and the Animal Care Center, Hubei Science and Technology Agency in China (SYXK 2013-0044). All experimental procedures in this study were performed according to the guidelines of the committee on the use and care of the laboratory animals in Hubei Province, China. All the animals were monitored throughout the study for any signs of adverse effects.

The target gene Cj0440c was down-regulated in the Cj0440c-inactivated mutants (SM and RM). The other differentially expressed genes in Cj0440c mutants (SM and RM) compared to their parental strains (S and R) were shown in Tables 2, 3. The relationship of these different genes was summarized in Figure 1.

A flagellin gene (flaA) and a gene (Cj1632c) encoding a putative periplasmic protein were up-regulated in SM as compared to S (indicated by red arrows in Figure 1). Among the down-regulated genes in SM (indicated by green arrows in Figure 1), 10 genes (flaB, flgE, flgE2, flgG, flgG2, flgH, flgI, flgK, flgL, and fliK) are possible involve in flagellar assembly; 2 genes (pseB and pseC) in carbohydrate metabolism; 2 genes (neuB₂ and neuC) in surface glycoprotein metabolism.

None of the genes were up-regulated genes were found in RM when compared the expression of different genes with R. Eleven down regulated genes in RM (indicated by blue arrows in Figure 1) are flagellar associated genes (flgD, fliD, flaG and flaB), a catalase encoding gene (katA) and four genes with unknown function (flgM, Cj1465, Cj0391, and Cj1242).

When submitted to STRING 9.05 and KEGG pathway analysis, the result showed that 10 flagellar genes were interacted with other down-regulated genes (pseB/C, neuB₂/C2 and Cj1026c) (Figure 1).

The transcriptional change of several representative genes detected in microarray was validated by qRT-PCR. The similar change of the selected genes was found both in microarray and qRT-PCR (Figure 2).

The microarray data obtained in this study have been deposited in the NCBI Gene Expression Omnibus database² and assigned accession numbers GSE49255 (RM&R) and GSE49255(SM&S).

As shown in Table 4, there was no significant difference between MIC of nine antimicrobial agents in Cj0440c mutants (SM and RM) comparing to their parental strains (S and R). Inactivation of Cj0440c did not affect antimicrobial susceptibility of C. jejuni.

The electron micrographs of all tested strains were shown in Figure 3. The results showed that parental strains (S and R) had long, spiral and complete flagella filaments in two sides (Figures 3A,C). However, SM had shorter filaments in only one side (Figure 3B). No filaments of RM were detected in RM (Figure 3D). These findings indicated that Cj0440c may affect the formation of flagella in C. jejuni.

Growth kinetics of Cj0440c mutants (SM and RM) and their parental strains (S and R) were determined in MH broth. No significant difference in growth rate was observed between SM and S. The RM exhibited slower growth rate compared to its parental R, however, the difference was not significant (Figure 4).

To determine the colonization capacity, broiler chickens were infected individually with four C. jejuni strains (S, R, SM and RM). All the strains were able to colonize in chicken intestinal tract, albeit at different rate. Comparing with the parental strains (S or R), the Cj0440c mutants (SM and RM) showed a significant reduction in colonization on 12 days’ post-inoculation (Figure 5).

When the two pairs of C. jejuni strains (SM&S and RM&R) were infected chickens with one pair at a time, Cj0440c mutants (SM and RM) exhibited lower colonization level compared to their parental strains (S and R) after 9 days’ post-inoculation (Figure 6).